Issued  January  17,  191:2. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  145. 

A.  D.  MELV1N,  CHIEF  01;  BUREAU. 


0  1 

0  ! 

1  ! 

0  I 
8! 
41 

I  3 
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4 


TRYPANOSOMA  AMERICANUM, 

iON  BLOOD  PARASITE  OF  AMERICAN  CATTLE. 


BY 


HOWARD   CRAWLEY, 
Junior  Zoologist,  Zpofagn'fal  Division. 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 
1912. 


Issued  January  17, 1912. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  145. 

A.  D.  MELVIN,  CHIEF  OF  BUREAU. 


TRYPANOSOMA  AWERICANUM, 

A  COMMON  BLOOD  PARASITE  OF  AMERICAN  CATTLE. 


BY 
HOWARD   CRAWLEY, 

Junior  Zoologist,  Zoological  Division, 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 
1912. 


THE  BUREAU   OF  ANIMAL  INDUSTRY. 


Chief:  A.  D.  MELVIN. 
Assistant  Chief:  A.  M.  FARRINGTON. 
Chief  Clerk:  CHARLES  C.  CARROLL. 

Animal  Husbandry  Division:  GEORGE  M.  ROMMEL,  chief. 
Biochemic  Division:  M.  DORSET,  chief. 
Dairy  Division:  B.  H.  RAWL,  chief. 

Inspection  Division:  RICE  P.  STEDDOM,  chief;  MORRIS  WOODEN,  R.  A.  RAMSAY, 
and  ALBERT  E.  BEHNKE,  associate  chiefs. 

Pathological  Division:  JOHN  R.  MOHLER,  chief. 
Quarantine  Division:  RICHARD  W.  HICKMAN,  chief. 
Zoological  Division:  B.  H.  RANSOM,  chief. 
Experiment  Station:  E.G.  SCHROEDER,  superintendent. 
Editor:  JAMES  M.  PICKENS. 

ZOOLOGICAL  DIVISION. 

Chief:  B.  H.  RANSOM. 

Assistant  Zoologists:  ALBERT  HASSALL,  HARRY  W.  GRAYBILL  and  MAURICE  C. 


Junior  Zoologists:  HOWARD  CRAWLEY  and  WINTHROP  D.  FOSTER. 

2 


LETTER  OF  TRANSMITTAL. 


U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY, 

Washington,  D.  C.,  August  31,  1911. 

SIR:  I  have  the  honor  to  transmit,  and  to  recommend  for  pub- 
lication in  the  bulletin  series  of  this  bureau,  the  accompanying  man- 
uscript entitled  "  Trypanosoma  americanum,  a  Common  Blood  Par- 
asite of  American  Cattle,"  by  Howard  Crawley,  of  the  Zoological 
Division. 

A  preliminary  description  of  this  trypanosome  was  prepared  in 

1909  by  Mr.  Crawley  and  published  hi  Bulletin  119  of  this  bureau. 

The  present  paper  presents  a  more  extended  study  of  the  organism, 

which  is  shown  to  be  harbored  by  about  75  per  cent  of  adult  cattle. 

Respectfully, 

A.  D.  MELVIN, 

Chief  of  Bureau. 
Hon.  JAMES  WILSON, 

Secretary  of  Agriculture. 


CONTENTS. 


Page. 

Introduction 5 

Re'sum6  of  literature 5 

Method  of  experimental  work 10 

Examination  of  cultures 10 

Seasonal  variation  in  number  of  trypanosomes  in  blood 13 

Effect  on  the  trypanosomes  of  keeping  blood  before  cultures  were  made  . .  16 

Results  obtained  with  measured  quantities  of  blood 18 

General  results  of  cultural  work 20 

Observations  on  fresh  material 21 

Attempts  to  discover  the  trypanosome  in  freshly  drawn  blood 23 

Evolution  of  the  trypanosomes  in  culture 24 

Movements  of  trypanosomes  in  cultures 29 

The  process  of  multiplication 30 

Motility  of  the  trypanosomes 31 

Observations  on  fixed  material 32 

Morphology  of  the  forms  in  the  blood 33 

Morphology  of  the  forms  in  culture 34 

Cytology  of  the  forms  in  culture 35 

Principal  characteristics  of  Trypanosoma  americanum 38 

Conclusions 38 

Bibliography 39 


ILLUSTRATIONS. 


[All  figures  made  from  camera  outlines  and  all  on  the  same  scale.] 

Page. 

FIG.  1  and  2.  Forms  of  Trypanosoma  americanum  from  centrifuged  blood 32, 33 

3.  Form  from  bouillon  culture  96  hours  old 34 

4  to  9.  Forms  from  bouillon  cultures  112  hours  old 35 

10.  Form  from  bouillon  culture  72  hours  old 36 

11  and  12.  Forms  from  bouillon  cultures  6  days  old 37 

13  to  15.  Forms  from  bouillon  cultures  10  days  old 37 

4 


TRYPANOSOMA  AMERICANUM,  A  COMMON  BLOOD 
PARASITE  OF  AMERICAN  CATTLE. 


INTRODUCTION. 

In  Bulletin  119  of  the  Bureau  of  Animal  Industry  there  was  de- 
scribed, under  the  name  of  Trypanosoma  americanum,  a  flagellated 
organism  which  appeared  in  bouillon  cultures  of  the  blood  of  American 
cattle.  At  the  time  of  writing,  although  but  7  cows  had  been  tested, 
trypanosomes  had  appeared  in  the  blood  cultures  of  all  of  them,  thus 
indicating  that  a  large  proportion  of  cattle  are  infected. 

The  study  therein  described  had  been  made  primarily  on  stained 
smears,  and  based  on  what  was  observed  in  these  it  was  stated  that 
the  earliest  stage  noted  was  a  round  or  oval  body,  occurring  in  clusters, 
and,  while  it  showed  a  kinetonucleus,  it  lacked  a  flagellum.  This  last 
observation  is  incorrect,  the  mistake  being  due  to  the  fact  that  in  these 
early  stages  the  flagellum  fails  to  stain.  A  more  extended  study, 
based  partly  on  fresh  material,  has  shown  that  the  organisms  in  the 
culture  tubes  are  always  flagellated.  It  is  desired  to  emphasize  the 
fact  that  this  observational  error  was  made,  since  the  precise  nature 
of  the  earliest  organism  to  appear  in  the  culture  tubes  is  a  matter  of 
some  moment,  bearing  as  it  does  on  the  problem  of  what  is  the  nature 
of  the  element  present  in  the  circulating  blood  of  the  cow.  It  may 
also  be  stated  here  at  the  outset  that  the  results  of  this  more  extended 
study  show  that  this  latter  undoubtedly  is  a  trypanosome  like  that 
present  in  the  culture  tubes,  because  trypanosomes  have  been  found 
in  the  blood  itself  within  a  few  hours  after  its  removal  from  the  cow. 

RESUME  OF  LITERATURE. 

There  is  a  very  considerable  literature  on  the  trypanosomes  of 
cattle,  and  a  number  of  species  have  been  described  as  occurring  in 
them.  It  will  not  be  necessary  here,  however,  to  do  more  than  con- 
sider that  which  bears  directly  on  the  problem  in  hand,  that  is,  which 
treats  of  trypanosomes  presumably  similar  to  Trypanosoma  ameri- 
canum.1 Some  of  this  literature  was  reviewed  in  Bulletin  119,  but 
one  paper  which  should  have  been  noted  was  overlooked,  and  several 
have  since  come  to  hand.  It  may,  however,  be  advisable  again  to 

1  References  to  literature  are  given  in  the  bibliography  at  the  end  of  this  bulletin. 


6  TBYPANOSOMA   AMEEICANUM,   A   BLOOD  PARASITE   OF   CATTLE. 

refer  to  Miyajima's  work,  since  this  is  the  point  of  departure  of  all  the 
studies  on  this  peculiar  trypanosome. 

The  organism  found  by  Miyajima  in  cultures  made  from  the  blood 
of  Japanese  cattle  is  apparently  the  same  as  that  found  in  the  United 
States.  Unfortunately  Miyajima  does  not  give  a  detailed  description 
of  his  parasite,  and  his  figures  are  not  particularly  clear.  They  show, 
however,  that  he  was  dealing  with  a  trypanosome  possessing  only  a 
very  short  undulating  membrane,  and  his  figure  10,  of  a  division 
form,  shows  two  granules  lying  at  the  same  level  and  in  the  anterior 
part  of  the  body.  From  this  it  may  be  concluded  that  the  Japanese 
form  has  the  characteristic  of  a  kinetonucleus  lying  very  close  to  the 
trophonucleus.  Further,  the  length,  given  as  five  times  the  diameter 
of  a  red  blood  cell,  or  say  30  microns,  is  in  general  agreement  with 
what  is  found  here. 

Martini  (1909),  working  at  Manila,  repeated  Miyajima' s  work,  and 
obtained  trypanosomes  in  bouillon  culture  tubes  inoculated  with  the 
blood  of  a  calf.  He  used  2  c.  c.  of  blood  to  10  c.  c.  of  bouillon.  In 
one  case  the  trypanosomes  appeared  in  33  hours.  As  a  rule,  however, 
the  period  was  from  43  to  48  hours.  The  tubes  were  kept  at  a  tem- 
perature of  26  to  27°  C.1  The  trypanosomes,  while  in  general  about 
the  size  of  Trypanosoma  lewisi,  showed  great  variation  in  length. 
Some  were  only  one  and  one-half  to  three  times  as  long  as  the  diameter 
of  a  red  blood  cell,  but  as  the  cultures  aged  they  became  longer,  and 
on  the  fifth  and  sixth  days  forms  were  present  20  to  25  tunes  the 
length  of  the  calf's  red  blood  cell.2  The  smallest  forms  were  morpho- 
logically the  same  as  the  largest.  The  anterior  end  of  the  flagellum 
showed  a  little  club-shaped  swelling.  The  kinetonucleus  lay  trans- 
versely, the  trophonucleus  longitudinally,  and  the  former  was  gener- 
ally in  front  of  the  latter.  The  parasites  grew  indifferently  in  acid 
or  alkaline  bouillon.  In  cultures  in  acid  media,  the  cytoplasm  con- 
tained chromatin  granules.  A  chronological  transition  was  not 
observed,  and  rudimentary  forms  were  always  to  be  found  alongside 
of  those  fully  developed.  Attempts  to  inoculate  monkeys  failed. 

Commenting  on  the  above,  there  can  be  no  question  that  Martini 
was  dealing  with  the  same  trypanosome  as  Miyajima,  which  is  also, 
in  all  probability,  the  one  which  appears  in  cultures  made  from  the 
blood  of  American  cattle. 

Wrublewski  (1908) 3  describes  a  trypanosome  found  in  the  blood 
of  the  Lithuanian  bison.  He  remarks  that  this  species  of  Bos  is  not  a 

i  Cultures  made  in  the  Zoological  Laboratory  of  the  Bureau  of  Animal  Industry  on  July  13, 1909,  showed 
trypanosomes  July  15,  the  mean  temperature  of  the  laboratory  for  this  period  being  27.4°  C. 

4  It  is  to  be  regretted  that  Martini  has  not  furnished  us  with  actual  measurements.  According  to  his 
own  figures,  the  calf  erythrocytes  range  in  diameter  from  4  to  6J  microns,  and  this  would  make  the  largest 
specimens  say  100  to  150  microns  long,  clearly  a  gross  exaggeration.  His  figure  12  shows  an  animal  40  to 
45  microns  long;  that  of  figure  14  is  perhaps  50,  and  these  are  the  largest  which  he  figures. 

3  This  paper  was  missed  at  the  time  of  writing  Bulletin  119. 


RESUME  OF  LITERATURE.  7 

favorable  subject  for  experimentation  and  hence  his  material  con- 
sisted of  smears  taken  from  dead  individuals.  The  trypanosomes  found 
ranged  in  length  from  30  to  50  microns.  The  posterior  end  is  elon- 
gated, but  terminates  bluntly.  In  some,  the  middle  portion  is  broad 
and  rounded  (compare  figs.  13  and  14,  this  bulletin) .  In  this  broad  por- 
tion are  to  be  found  the  kinetonucleus  and  the  trophonucleus,  the  for- 
mer in  front  of  the  latter.  The  root  of  the  flagellum  is  broad  and  may 
show  a  knoblike  expansion.  The  free  end  of  the  flagellum  is  also 
knobbed.  Around  the  trophonucleus  are  masses  of  deeply  staining 
granules,  and  granules  may  also  be  found  scattered  throughout  the 
entire  body. 

In  some  cases  Wrublewski  obtained  blood  sufficiently  fresh  to 
show  living  trypanosomes.  They  are  very  energetic,  and  in  hanging 
drops  dash  through  the  red  cells  with  extraordinary  velocity,  hurling 
these  to  one  side.  The  parasite  moves  with  the  flagellum  in  front. 
The  movement  is  rotatory. 

Vladimiroff  and  Yakimoff  (1908)  name  this  trypanosome  Trypano- 
soma  wrublewski. 

Of  the  several  bovine  trypanosomes  described  as  occurring  in  the 
circulating  blood,  this  comes  by  far  the  closest  to  T.  americanum. 
Wrublewski's  figures  3,  4,  and  5  would  answer  for  specimens  of  the 
American  species.  The  most  noteworthy  distinction  is  in  the  mo- 
tility,  T.  americanum  rarely  showing  a  rapid  translatory  movement. 
There  is  no  especial  reason  why  the  two  might  not  be  the  same,  the 
parasite  properly  belonging  to  the  bison  and  transferred  from  it  to 
domesticated  cattle  by  natural  means.  The  fact  that  the  parasite 
is  apparently  quite  abundant  in  the  circulating  blood  of  the  bison, 
and  very  scarce  in  that  of  ordinary  cattle,  is  no  argument  against 
their  identity.  Moreover,  the  sluggishness  of  T.  americanum  in  cul- 
ture tubes  is  no  criterion  as  to  how  it  may  behave  in  the  circulating 
blood,  regarding  which  there  is,  unfortunately,  no  information  at 
hand. 

Recent  studies  have  shown  trypanosomes  to  be  present  in  a  large 
proportion  of  cattle  in  Germany,  or  at  least  capable  of  appearing  in 
cultures  made  from  their  blood. 

Knuth  and  Rauchbaar  (1910)  tested  17  adult  cattle  and  2  calves, 
and  found  10  of  the  adults  to  be  parasitized.  Later,  out  of  a  lot  of  9 
cattle,  6  were  found  infected. 

Knuth,  Rauchbaar,  and  Morgenstern  (1910)  found  trypanosomes 
hi  culture  tubes  from  7  out  of  25  cattle.  They  note  the  presence  in 
the  tubes  of  developmental  forms  and  of  agglomerations. 

Behn  (1910  /?),  culturing  cattle  blood  in  the  same  manner,  found 
flagellated  forms  on  the  second  day.  Furthermore,  in  smears  made 
from  cultures  1  to  2  days  old,  he  found  round  bodies,  in  many  cases 


8  TRYPANOSOMA   AMERICANUM,    A   BLOOD  PARASITE   OF   CATTLE. 

with  alveolar  protoplasm,  which  stained  an  intense  blue  with  Giemsa. 
These  showed  one  or  more  large,  round  nuclei,  variable  in  size  and 
staining  red.  The  bodies  varied  in  size,  the  largest  being  nearly  as 
large  as  a  leucocyte.  Some  were  free,  others  inclosed  in  white  blood 
cells.  Behn  appears  to  consider  that  the  intracellular  forms,  growing, 
break  open  the  leucocytes  and  escape.  He  bases  this  view  on  the 
fact  that  in  smears  from  cultures  2  days  old  the  bodies  then  had 
attached  to  them  the  remnants  of  disintegrated  leucocytes.  He  pro- 
poses the  query  as  to  whether  he  is  dealing  with  a  case  of  phaf  ocy- 
tosis  or  of  an  evolution  of  the  parasites  within  the  leucocytes,  and 
promises  a  more  detailed  study. 

No  bodies  such  as  the  above  have  been  seen  in  smears  made  from 
American  cattle,  and  it  is  perhaps  possible  that  Behn  was  dealing  with 
moribund  or  dead  leucocytes. 

The  same  author  (Behn,  1910  a)  refers  to  the  seven  animals  men- 
tioned by  Knuth,  Rauchbaar,  and  Morgenstern.  He  states  that 
whereas  the  cultures  from  these  were  always  positive  from  the  begin- 
ning of  August  until  the  end  of  September,  after  cool  weather  set  in 
they  no  longer  developed  trypanosomes.  One  of  these  cows  failed 
to  show  parasites  after  September  11.  But  in  a  smear  of  her  blood 
taken  August  8  there  was  found  a  single  trypanosome,  remarkable 
for  its  unusual  breadth. 

Behn  gives  the  following  measurements : 

Microns. 

Posterior  end  to  middle  of  kinetonucleus 13 

Middle  of  kinetonucleus  to  posterior  end  of  trophonucleus 4 

Trophonucleus 2 

Anterior  end  of  trophonucleus  to  anterior  end 24 

Length  of  body 43 

Length  of  free  flagellum 12 

Total  length 55 

Maximum  width 12 

The  trypanosome  had  the  body  sharply  bent.  The  cytoplasm 
showed  clear  spaces  and  abundant  granules.  The  granules  were  al- 
most all  of  the  same  size  and  stained  reddish  to  a  blue-violet.  Around 
the  nucleus  the  cytoplasm  was  freer  from  granules  than  elsewhere. 
The  kinetonucleus  stained  a  black  violet,  and  lay  hi  a  clear  region. 
The  nucleus  lay  transversely,  filling  the  entire  width  of  the  body. 
It  stained  pale  red. 

Continuing  his  studies  Behn  (1910  ;•)  inoculated  a  calf  with  blood 
from  a  cow  whose  blood  gave  positive  cultures,  but  was  negative  to 
direct  examination.  Eleven  days  later  the  blood  of  this  calf  was 
positive  to  direct  examination.  For  the  first  few  days  the  trypano- 
somes seen  were  of  the  Trypanosoma  franki  type,  but  later  they  took 
on  the  appearance  of  T.  tJieileri.  Cultures  made  from  the  blood  of  this 
calf,  after  the  trypanosomes  had  appeared,  were  positive. 


RESUME   OF   LITERATURE. 


Knuth  (1910)  tested  by  the  cultural  method  the  blood  of  41  cattle, 
with  the  following  results : 


, 

Total 
number 
tested. 

Positive 
for 
trypano- 
somes. 

Adult  cattle  

31 

21 

Young  cattle                    .             

7 

1 

Sucking  calves  

3 

0 

Schmitt  (1910)  also  found  trypanosomes  of  the  iheileri  type  in  a 
Pomeranian  cow  suffering  from  Texas  fever.  These  were  found  in  the 
blood,  were  rare,  and  were  present  for  10  days. 

Sergent,  E.  and  E.,  (1911)  made  cultures  of  the  blood  of  82  cattle 
killed  at  the  abattoir  at  Algiers.  Trypanosomes  were  obtained  nine 
tunes.  Division  forms  were  abundant,  and  subcultures  were  made. 
The  medium  used  was  bouillon. 

Delanoe  (1911),  at  Alfort,  made  cultures  of  cattle  blood,  using  3  c.  c. 
of  defibrinated  blood  in  10  c.  c.  of  bouillon.  Six  out  of  the  ten  animals 
tested  gave  positive  results,  but  not  all  of  the  cultures  made  from 
parasitized  animals  were  positive,  indicating  the  great  rarity  of  the 
trypanosomes  in  the  blood.  The  elements  found  in  the  cultures  were 
crithidia-like,  averaged  50  microns  in  total  length,  and  the  free  tip  of 
the  flagella  was  broadened.  Chromatoid  granules  appeared  in  the 
cytoplasm  only  in  the  old  cultures.  In  a  subculture  in  Nicolle's 
medium,  there  were  seen  very  small  forms  without  flagella,  flagel- 
lated elements  35  microns  long,  and  trypanosomes  with  a  posterior 
kinetonucleus. 

Knuth  confirms  Behn  in  the  respect  that,  after  the  onset  of  cold 
weather,  animals  having  given  positive  results  cease  to  do  so.  He 
suggests  that  this  may  be  due  either  to  the  disappearance  of  the 
insect  carriers  or  to  the  failure  of  the  trypanosomes  to  grow  in  the 
tubes  in  cold  weather.  Finally,  by  private  correspondence,  Knuth 
has  been  advised  that  blood  cultures  of  cattle  have  been  made  in  a 
number  of  laboratories  in  Germany,  Denmark,  and  Sweden,  with 
the  subsequent  finding  of  trypanosomes. 

Dudukalov  and  Dudukalova  (1910)  report  the  results  of  an  experi- 
mental study  on  the  trypanosome  of  the  cow.  The  culture  media 
used  were  (a)  bouillon;  (b)  equal  parts  of  bovine  blood  and  0.8  per 
cent  NaCl  solution;  l  (c)  agar.  Each  tube  was  inoculated  with  a 
few  drops  of  blood.  After  3  to  4  days  there  appeared  in  the  tubes 
round  forms  of  the  parasite,  about  the  size  of  leucocytes,  in  many 
cases  occurring  in  great  clusters.  On  the  sixth  to  seventh  days 
elongated  bodies  appeared  among  these  round  bodies,  and  on  the 
tenth  to  twelfth  days  there  were  present  great  numbers  of  motile 

i  The  text  says  0.08  NaCl  solution,  doubtless  a  printer's  error. 
10414°— Bull.  145—12 2 


10        TRYPANOSOMA  AMERICANUM,   A   BLOOD  PARASITE   OF   CATTLE. 

trypanosomes  provided  with  long  flagella.  After  2  to  3  weeks  the 
trypanosomes  were  present  in  enormous  numbers  and  were  of 
various  sizes  and  forms.  They  remained  alive  in  the  tubes  for  2  to 
4  months.  Subinoculations  were  made  and  carried  to  the  fourth 
generation.  The  optimum  temperature  for  the  cultures  is  10  to 
15°  R. 

With  reference  to  the  above  papers,  the  observation  of  the  German 
authors  that  the  trypanosomes  no  longer  develop  in  the  tubes  after 
the  onset  of  cold  weather  is  confirmed  by  the  studies  made  here, 
as  will  be  pointed  out  later.  As  to  that  of  the  Dudukalovs,  it  would 
be  of  interest  to  know  if  the  round  bodies  first  found  were  flagellated. 
If  so,  the  Russian  authors  were  dealing  with  a  process  altogether 
like  that  displayed  by  Trypanosoma  americanum.  The  more 
leisurely  development  can  be  explained  by  the  rather  low  temperature 
to  which  their  cultures  were  subjected.  It  should  also  be  noted 
that  the  figures  accompanying  their  original  article — the  re'sume' 
given  above  having  been  taken  from  a  review  in  a  less  difficult  lan- 
guage than  Russian — show  that  they  were  dealing  with  a  trypanosome 
morphologically  identical  with  T.  americanum.  On  the  other  hand, 
it  was  not  possible  to  get  the  American  trypanosome  to  develop  in 
salt  solution,  even  though  an  equal  bulk  of  blood  had  been  added. 

Stockman  (1910)  found  trypanosomes  in  the  blood  of  6  out  of 
10  cattle  which  had  been  inoculated  with  Piroplasma  to  immunize 
against  piroplasmosis.  In  one  case  the  trypanosomes  were  present 
for  eight  days;  in  the  other  five  for  only  a  day  or  two.  The  try- 
panosomes were  morphologically  indistinguishable  from  T.  iheileri, 
and  would  not  grow  in  culture  tubes. 

Stockman  makes  no  comment,  but  there  is  no  reason  for  regarding 
what  he  found  as  anything  but  T.  iheileri,  rendered  more  abundant 
as  a  result  of  the  weakened  condition  of  the  cattle. 

METHOD  OF  THE  EXPERIMENTAL  WORK. 

The  procedure  followed  was  to  prepare  cultures  of  the  blood  of  the 
cattle,  and  to  examine  fresh  or  fixed  preparations  made  from  these. 
The  method  of  drawing  the  blood,  making  the  cultures,  etc.,  has 
been  described  in  Bulletin  119,  and  need  not  be  repeated  here.  The 
study  falls  easily  into  two  parts,  (1)  that  of  the  cultures  themselves, 
or  what  may  be  called  the  experimental  work,  and  (2)  that  made 
with  the  microscope  on  fresh  or  stained  material.  The  work  with 
the  cultures  will  be  detailed  first. 

EXAMINATION  OF  CULTURES. 

In  all,  several  hundred  cultures  were  made.  The  media  used 
were  beef  bouillon,  mutton  bouillon,  bouillon  made  from  extract  of 
meat,  and  salt  solution.  In  some  cases  the  cultures  were  made  the 


EXAMINATION   OF   CULTURES.  11 

same  day  as  the  blood  was  drawn;  in  others  an  interval  of  from 
one  to  several  days  was  allowed  to  elapse,  the  blood  meanwhile 
being  kept  either  in  the  ice  chest,  in  the  laboratory,  or  in  the  incubator. 
Further,  the  blood  was  sometimes  used  in  measured  quantities; 
in  other  cases  there  was  merely  added  to  the  medium  what  was 
considered  to  be  a  sufficient  amount. 

At  first  the  cultures  were  in  some  cases  placed  in  the  incubator, 
but  the  behavior  of  Typanosoma  americanum  is  here  the  same  as 
that  of  any  other  trypanosome  in  culture,  and  cultures  kept  at 
incubator  temperature  quickly  deteriorate.  The  explanation  has 
been  advanced  that  the  preference  of  a  "  cultural "  form  for  a  moderate 
temperature  is  due  to  the  fact  that  the  biting  arthropod  which 
removes  it  from  the  mammalian  host  has  the  temperature  of  the 
surrounding  air.  Without  dogmatizing  as  to  whether  or  not  this 
may  be  true,  it  is  perfectly  evident  that  the  reason  T.  americanum 
can  not  live  in  cultures  in  the  incubator  is  because  these  cultures 
spoil.  Apparently,  without  reference  to  the  presence  or  absence  of 
bacteria,  the  hemoglobin  leaches  out  of  the  red  cells  and  goes  into 
solution  in  the  medium,  and  there  can  be  no  doubt  that  the 
liquids  in  the  tube  undergo  profound  chemical  changes.  Hence, 
in  a  very  great  majority  of  cases,  the  cultures  were  kept  at  room 
temperature,  which  varied  from  20  to  27.5°  C.,  according  to  the 
time  of  year. 

In  the  greater  number  of  cultures  the  medium  used  was  neutral 
beef  bouillon,  prepared  in  the  laboratory.  The  tubes  were  charged 
the  same  day  the  blood  was  drawn,  and  the  quantity  of  blood, 
while  not  measured,  varied  from  1  to  4  c.  c.  The  quantity  of  bouillon 
was  not  measured  accurately,  but  ranged  from  3  to  6  c.  c.  It  was 
mainly  from  such  cultures  as  these  that  the  material  used  in  the 
microscopical  studies  was  obtained. 

It  is  not  necessary  to  tabulate  the  results  obtained  from  this  lot  of 
cultures,  as  such.  The  more  interesting  results  were  obtained 
from  those  experiments  in  which  the  quantity  of  blood  was  measured 
and  from  those  carried  out  with  reference  to  the  season  of  the  year. 
The  former  are  necessarily  an  entirely  different  set  from  those  in 
which  the  blood  was  not  measured,  and  the  latter  set  was  made 
up  of  both  measured  and  unmeasured  cultures.  Tables  for  both  of 
these  sets  of  tubes  are  given  later. 

It  is  evident  that  in  order  to  determine  the  number  of  days  required 
for  the  trypanosomes  to  appear  in  a  tube,  the  tube  must  be  examined 
daily  from  the  time  of  making  until  the  trypanosomes  appear. 
This  was  done  for  53  tubes,  taken  throughout  the  year,  and  it  was 
found  that  the  average  time  required  was  3£  days.  It  is  understood 
that  this  means  the  time  required  for  the  presence  of  trypanosomes 
on  the  top  of  the  column  of  red  cells  to  be  determined  or  at  least 


12        TRYPANOSOMA   AMERICANUM,   A  BLOOD  PARASITE   OF   CATTLE. 


suspected,  merely  by  the  use  of  a  hand  lens,  although  in  almost 
all  cases  this  diagnosis  was  confirmed  by  the  use  of  the  microscope. 
This  average  of  3£  days  is  based  on  cultures  made  the  day  the  blood 
was  drawn  and  which  were  kept  at  room  temperature. 

In  all  30  animals  were  used,  and  64  separate  tests  were  made  of 
their  blood.  Of  these  animals  27  were  yearlings  or  adults  and  3  were 
young  calves.  The  calves,  one  of  which  was  but  1  day  old,  were  all 
negative.  The  mother  of  the  1-day  calf,  tested  at  the  same  time, 
was  positive,  an  indication  that  trypanosomes  can  not  pass  the 
placenta. 

Of  the  27  adult  animals,  7  gave  negative  results,  hence  74  per  cent 
were  infected.  The  actual  figure,  however,  is  probably  higher  than 
this,  since  a  single  test  may  be  negative  merely  by  accident,  and  of 
these  7  cattle,  5  were  tested  but  once.  Moreover,  as  will  be  shown, 
the  time  of  year  must  be  taken  into  account. 

The  following  were  six  of  the  cattle  which  proved  negative,  with 
the  month  when  the  test  was  made;  the  seventh,  No.  685,  is  dealt 
with  below:  Nos.  666  and  668,  tested  in  March;  No.  667,  tested  in 
April;  No.  739,  tested  in  May  and  also  in  October;  No.  536,  tested  in 
July;  and  No.  738,  tested  in  October. 

By  October  the  trypanosomes  have  become  much  less  abundant  in 
the  blood,  and  an  animal  negative  in  that  month  might  readily  have 
been  positive  in  July  or  August.  Nevertheless,  certain  cattle  do  not 
harbor  the  trypanosomes,  as  the  following  history  of  cow  No.  685 
shows : 


Date. 

Number  of 
cultures 
made. 

Result. 

1909. 
July  13  

5 
4 
3 
Received 
vein  20  c. 
cow  No. 
carry  try 
3 
3 
3 
6 

All  negative. 
Do. 
Do. 
in    the   jugular 
c.  of  blood  from 
689,  known  to 
sanosomes. 
All  negative. 
Do. 
Do. 
Do. 

July  20  

Aug.  18  

Aug.  26  

Aug.  30  

Sept.  2          

Sept.  8  

Nov.  17                 .             

This  is  apparently  a  case  of  natural  immunity.  The  inoculation 
might  have  failed,  but  the  constant  negative  findings  for  five  months, 
including  the  greater  part  of  the  hot  season,  show  that  the  animal  was 
able  to  resist*  natural  infection.  There  was  nothing  in  the  history  of 
this  animal  to  differentiate  it  from  any  other,  and  it  was  in  precisely 
the  same  environment  as  all  of  the  other  cattle  at  the  Experiment 
Station.  In  fact,  both  it  and  No.  689,  from  which  it  was  inoculated, 
were  kept  for  a  part  of  the  summer  in  the  same  pen. 


EXAMINATION   OF   CULTURES.  13 

SEASONAL   VARIATION    IN   NUMBER   OF   TRYPANOSOMES    IN    BLOOD. 

The  seasonal  variation  in  the  abundance  of  trypanosomes  is  very 
well  shown  by  the  history  of  cow  No.  218,  as  follows: 

Appearance  of  trypanosomes  in  cultures  from  cow  No.  218. 


No.  of  culture. 

Date. 

Quantity 
of  blood. 

Time  re- 
quired for 
trypanosomes 
to  appear. 

Remarks. 

244                        ... 

Aug.  18 

Not  meas- 

4 days  

245 

do 

ured.* 
do 

5  days 

294 

Aug.  31 

do  

6  days  . 

295  

...do  

...do  

4  days  

Do. 

299 

Sept.    1 

.do.... 

5  days. 

303  

Sept.    2 

...do.... 

do  

Blood  kept  on  ice  3  days 

309 

Sept.    3 

.do.... 

4  days 

330 

Sept.    8 

do  

3  days  

331 

do  .  .. 

do     . 

2  days 

334 

do  

.do  

..     .do  

420  

Nov.  17 

1  c.  c  

Negative  

421 

do  .    . 

2c.  c 

do.  . 

422  

...do  

3c.  c  

...do... 

423  

...do  

4c.  C  

do  

Contaminated. 

424  
425 

...do  
do  . 

5c.  C  

G  c.  c 

do  
6  days 

426 

...do  

7  c.  c    

Negative  

427 

do 

8c  c 

6  days 

428  

...do  

9c.  c  

do  

429  
436  

...do  
...do  

10  c.  c  
1  c.  c  

do  
Negative  

438              

...do  

3  c.  c    .  . 

.do  

440  

...do.... 

5c.  c  

6  days  

441 

.do... 

6c.  c 

Negative. 

442  

...do.... 

7c.  C  

10  days  

443 

do.. 

8c.  c 

Negative 

445  

...do.... 

10  c.  c... 

5  days.  .   . 

1  From  1  to  4  c.  c.,  probably  averaging  3  c.  c. 

From  this  table  we  see  that  in  August  and  September  the  cultures 
were  all  positive,  and  some  showed  trypanosomes  as  early  as  the 
second  day.  On  the  other  hand,  of  the  17  cultures  made  in  November, 
only  7  were  positive,  with  5  days  as  the  minimum  time.  Moreover, 
while  the  average  quantity  of  blood  used  in  August  and  September 
was  probably  about  3  c.  c.,  and  in  no  case  exceeded  4.  c.  c.,  the  least 
quantity  to  develop  trypanosomes  in  November  was  5  c.  c.,  and  one 
each  of  the  cultures  containing  7  and  8  c.  c.,  respectively,  were  negative. 

In  the  case  of  cow  No.  697,  from  which  many  cultures  were  made, 
it  was  found  that  a  larger  proportion  of  tubes  were  positive,  and 
trypanosomes  appeared  more  quickly  in  spring  and  summer  than 
they  did  in  the  autumn. 

The  table  following  shows  all  the  cases  where  the  time  of  appearance 
of  the  trypanosomes  was  determined.  It  gives  the  number  of  cultures 
so  determined,  the  maximum  and  minimum  time  required  for  the 
trypanosomes  to  appear,  in  days,  and  the  average  of  the  whole  number. 
There  is  also  given  the  mean  temperature  of  each  month,  as  obtained 
at  the  Washington,  D.  C.,  station  of  the  United  States  Weather 
Bureau,  and  the  mean  temperature  of  the  zoological  laboratory. 


14        TBYPANOSOMA  AMERICANUM,   A   BLOOD   PARASITE    OF   CATTLE. 
Time  of  appearance  of  trypanosomes  in  laboratory  cultures,  by  months. 


Month. 

Number 
of  tubes. 

Time  of  appearance. 

Temperature. 

Maximum. 

Minimum. 

Mean. 

Washing- 
ton. 

Labora- 
tory. 

1909. 
April  

15 
12 
1 
11 
13 
6 
12 
15 

8 
15 

Days. 
4 

f 

6 
6 
8 
9 

6 
4 

Days. 
3 

i1 

2 
3 

2 
7 
3 

2 
3 

Days. 
3.3 
3.0 
3.0 
3.3 
4.4 
3.3 
7.2 
6.2 

4.1 
3.3 

°F.     "C. 

54.2=12.3 
64.4=18.0 
73.4=23.0 
74.7=23.7 
73.0=22.8 
66.4=19.1 
53.2=11.8 
50.8=10.4 

51.2=10.7 
57.9=14.4 

°F.    °C. 
72.5=22.5 
74.3=23.5 
78.7=25.9 
81.5=27.5 
79.1=26.2 
75.2=24.0 
70.4=21.4 
71.7=22.1 

74.4=23.6 
74.1=23.4 

May  

June  

July  

August      ..      .        

September 

October  

November  

1910. 
March  

April  

The  reason  why  the  laboratory  temperature  is  always  higher  than 
that  of  the  open  air  is  doubtless  obvious  enough,  although  attention 
may  be  called  to  the  fact  that  in  hot  weather  the  closing  of  the  labora- 
tory windows  at  4.30  p.  m.  prevents  any  such  nightly  fall  of  tempera- 
ture as  is  usual  in  the  open.  The  temperature  as  recorded  by  the 
Weather  Bureau,  although  taken  in  the  city  itself,  may  be  assumed  to 
correspond  to  that  of  the  Experiment  Station  of  the  Bureau  of 
Animal  Industry,  the  home  of  the  cattle.  Accordingly,  the  one 
column  shows  the  fluctuations  of  temperature  to  which  the  animals 
harboring  the  trypanosomes  were  subjected,  the  second  that  in  which 
the  tubes  were  kept.  The  former  can  hardly  be  supposed  to  have  any 
influence,  since  a  mammal  maintains  its  own  temperature  without 
reference  to  that  of  the  surrounding  air.  Therefore  in  making  com- 
parisons between  the  rates  of  growth  of  the  trypanosomes  at  different 
times  of  the  year,  it  is  the  temperature  of  the  laboratory  which  should 
be  taken  into  account  and  not  that  of  the  open  country. 

In  the  following,  taken  from  the  table  given  above,  the  months  are 
in  the  first  two  cases  grouped  with  reference  to  uniformity  of  tempera- 
ture and  in  the  last  with  reference  to  the  time  required  for  the  cultures 
to  develop. 

1.   Variation  in  time  of  appearance  of  trypanosomes  although  monthly  temperatures  are 

closely  uniform. 


Month. 

Labora- 
tory tem- 
perature. 

Mean 
time  of 
appear- 
ance. 

April,  1909 

'F. 
72.5 

Days. 
3.3 

November,  1909  

71.7 

6.2 

October,  1909 

70.4 

7.2 

EXAMINATION    OF   CULTURES. 
2.    Uniformity  in  time  of  appearance  of  trypanosomes  in  spring  months. 


15 


Month. 

Labora- 
tory tem- 
perature. 

Mean 
time  of 
appear- 
ance. 

March,  1910... 

°F. 

74  1 

Days. 
4.1 

April,  1910  

74.4 

3.3 

May,  1909.. 

74.3 

3.0 

3.   Uniformity  in  time  of  appearance  of  trypanosomes  although  variation  in  monthly 
temperatures  is  considerable. 


Month. 

Labora- 
tory tem- 
perature. 

Mean 
time  of 
appear- 
ance. 

April,  1909... 

°F. 
72.5 

Days. 
3.3 

May,  1909 

74.3 

3  0 

July,  1909..                                                         .  . 

81.5 

3.3 

September,  1909 

75.2 

3.3 

April,  1910  

74.1 

3.3 

In  the  first  group  it  is  seen  that  although  the  temperatures  are  very 
close,  the  time  required  hi  one  case  is  more  than  double  that  of 
another.  In  the  second  group  the  maximum  difference  in  tune  is 
only  1.1  days,  but  here  the  comparison  is  between  the  three  spring 
months.  In  the  third,  although  the  times  are  so  near  alike  that  the 
difference  is  negligible,  there  is  a  maximum  difference  of  9°  F.  in  the 
temperature. 

In  the  statement  below  the  entire  time  during  which  the  study 
was  being  carried  on  is  divided  according  to  the  three  seasons — spring, 
summer,  and  autumn.  The  average  time  required  for  the  cultures 
to  develop  during  each  season  is  computed,  due  weight  being  given 
to  the  number  of  cultures  used,  and  the  mean  temperature,  both  of 
the  open  country  and  the  laboratory,  are  given. 

Time  of  appearance  of  trypanosomes  according  to  seasons. 


Season. 

Time  of 
appear- 
ance. 

Mean  temperature. 

Outside. 

In  lab- 
oratory. 

Spring  .                .     .        .          

Days. 
3.39 
3.86 
6.04 

°F. 
56.9 
73.7 
56.8 

°F. 
73.8 
79.8 
72.4 

Summer  

A  utumn       .                            .                        

There  is  seen  here  a  considerable  seasonal  difference  in  the  time 
required  for  the  trypanosomes  to  appear  in  the  tubes.  This  is  taken 
to  be  an  index  to  their  abundance.  The  mode  of  examination  of  the 
cultures  was  throughout  much  the  same,  namely,  a  preliminary 
observation  of  the  top  of  the  column  of  blood  cells  with  a  hand  lens, 
followed,  when  necessary,  by  the  examination  of  a  drop  of  the  culture 


16        TRYPANOSOMA   AMERICANUM,   A  BLOOD   PARASITE   OF   CATTLE. 


under  the  microscope.  The  latter  was  undertaken  only  when  the 
former  left  the  question  as  to  the  presence  or  absence  of  trypanosomes 
in  doubt. 

As  we  shall  see  later,  multiplication  of  the  trypanosomes  begins  as 
soon  as  the  blood  containing  them  is  removed  from  the  cow.  It 
therefore  seems  reasonable  to  regard  their  discovery  in  the  culture 
tubes  by  means  of  the  procedure  indicated  above  as  evidence  of 
greater  abundance  rather  than  a  mere  rapid  multiplicative  rate. 
Hence  it  is  believed  that  these  figures  furnish  satisfactory  evidence  to 
show  that  whereas  the  trypanosomes  are  less  abundant  in  autumn 
than  hi  spring  or  summer,  this  difference  is  independent  of  tempera- 
ture. The  figure  for  spring  is  also  lower  than  that  for  summer,  but 
this  difference  is  not  large  enough  to  warrant  any  conclusions. 

EFFECT  ON  THE  TRYPANOSOMES  OF  KEEPING  BLOOD  BEFORE  CULTURES 

WERE    MADE. 

The  following  table  gives  the  results  obtained  when  the  blood  was 
kept  one  or  more  days  before  the  cultures  were  made.  The  several 
columns  show,  in  order,  the  number  of  the  cow;  date  culture  was 
made;  date  blood  was  drawn;  interval  in  days;  number  of  tubes  used; 
number  of  tubes  giving  positive  results;  and  place  where  blood  was 

kept. 

Result  of  examination  of  laboratory  cultures  for  trypanosomes. 


No.  of  cow. 

Culture 
made. 

Blood 
drawn. 

Interval 
(days). 

Number 
of 
tubes. 

Number 
of  posi- 
tive 
cases. 

Place  blood  was 
kept. 

688 

1909. 
Apr.     2 

1909. 
Mar.  31 

2 

3 

0 

On  ice. 

696  

May     7 

May     2 

5 

0 

Do. 

688 

Aug.  19 

Aug.  17 

2 

4 

0 

In  laboratory. 

689  

Aug.  27 

Aug.  26 

1 

2 

0 

Do. 

218 

Aug.  27 

Aug.  26 

1 

2 

0 

Do. 

689  

Aug.  28 

Aug.  26 

2 

2 

0 

Do. 

218 

Aug.  28 

Aug.  26 

2 

2 

0 

Do. 

689..          .              

Aug.  29 

Aug.  26 

3 

2 

0 

Do. 

218  . 

Aug.  29 

Aug.  26 

3 

2 

0 

Do. 

689..              . 

Aug.  30 

Aug.  26 

4 

2 

0 

Do. 

218 

Aug.  30 

Aug.  26 

4 

2 

0 

Do. 

689  

Aug.  31 

Aug.  26 

2 

0 

Do. 

218 

Aug.  31 

Aug.  30 

1 

2 

0 

In  incubator. 

218  

Aug.  31 

Aug.  30 

1 

2 

2 

On  ice. 

218  

Sept.    1 

Aug.  30 

2 

2 

0 

In  incubator. 

218 

Sept.    1 

Aug.  30 

2 

2 

1 

On  ice. 

218  

Sept.    2 

Aug.  30 

3 

2 

0 

In  incubator. 

218 

Sept.    2 

Aug.  30 

3 

2 

1 

On  ice. 

218  

Sept.    3 

Aug.  30 

4 

2 

0 

In  incubator. 

218...   . 

Sept.    3 

Aug.  30 

4 

2 

1 

On  ice. 

696  

Sept.   4 

Sept.    3 

1 

2 

0 

In  laboratory. 

697.. 

Sept.   4 

Sept.    3 

1 

2 

2 

Do. 

696  

Sept.    5 

Sept.    3 

2 

2 

0 

Do. 

697. 

Sept.   5 

Sept.    3 

2 

2 

0 

Do. 

696 

Sept.   6 

Sept.    3 

2 

2 

0 

Do. 

697... 

Sept.   6 

Sept.    3 

3 

2 

1 

Do. 

218 

Sept.  14 

Sept.  13 

1 

2 

0 

On  ice. 

218  

Sept.  15 

Sept.  13 

2 

2 

0 

Do. 

218 

Sept.  16 

Sept.  13 

2 

2 

0 

Do. 

688... 

Nov.    4 

Nov.    3 

1 

4 

1 

In  incubator. 

696... 

Nov.    4 

Nov.    3 

1 

4 

0 

Do. 

697  

Nov.    4 

Nov.    3 

1 

4 

0 

Do. 

730. 

1910. 
Mar.  11 

1910. 
Nov.    9 

2 

2 

2 

On  ice. 

730  

Mar.  29 

Nov.  16 

13 

4 

0 

Do. 

EXAMINATION   OF   CULTURES. 


17 


The  following  data,  taken  from  above,  give  the  place  where  the 
blood  was  kept,  the  length  of  time  kept,  the  total  number  of  cultures 
made,  and  the  number  which  developed  trypanosomes: 

Blood  kept  on  ice. 


Time 

Culture 

Positive 

kept. 

tubes. 

cases. 

1  day  .  . 

4 

2 

2  days. 

9 

3 

3  days. 

4 

1 

4  days. 

2 

1 

5  days. 

5 

0 

13  days. 

4 

0 

Blood  kept  in  laboratory. 


Time 

Culture 

Positive 

kept. 

tubes. 

cases. 

1  day  

8 

2 

2  days  — 

12 

0 

3  days  — 

8 

1 

4  days  — 

8 

0 

5  days  — 

2 

0 

Blood  kept  in  incubator. 


Time 

Culture 

Positive 

kept. 

tubes. 

cases. 

Iday  

14 

1 

2  davs  — 

2 

0 

3  days  — 

2 

0 

4  days  — 

4 

0 

Thus,  eleven  cultures  developed  trypanosomes,  and  the  following 
shows  the  time  required  in  each  case  for  the  organisms  to  appear  in 
the  tubes: 

Blood  on  ice. 


Time  kept. 

Time  of 
appearance. 

Remarks. 

1  day  — 

'2  days.  .  . 
3  days. 

a  
b 

(idays  
3  days  
5  days  
4  days  
•M  days... 
5  days  
4  days  

Good  growth. 
Do. 
Do. 

Poor  growth. 
Moderate  growth. 

a  
b  
c..  .  . 

4  days. 

Me: 

in.. 

4J  days  — 

10414°— Bull.  145—12- 


18        TBYPANOSOMA  AMERICANUM,   A   BLOOD  PARASITE   OF   CATTLE. 

Blood  in  laboratory. 


Time  kept. 

Time  of 
appearance. 

Remarks. 

t  j          fa. 

3  days. 

Very  poor  growth. 

iday....$ 

4-3  days. 

3  days.. 

moderate  growth. 
Not   examined   until 

15th  day,  when  it 
was  positive. 

Blood  in  incubator. 


Time  kept. 

Time  of 
appearance. 

Remarks. 

1  day. 

12  days 

When  the  blood  was  kept  in  the  ice  chest  at  a  temperature  of  about 
16°  C.  for  1  to  5  days,  only  7  out  of  24  cultures  developed,  or  29  per 
cent.  For  blood  kept  in  the  laboratory  the  figure  is  3  out  of  38,  or  8 
per  cent.  With  blood  kept  hi  the  incubator,  but  one  culture  out  of 
22  produced  trypanosomes.  This  shows  that  keeping  the  blood  be- 
fore the  cultures  are  made  has  a  destructive  influence  on  the  trypano- 
somes. 

As  to  the  time  required  for  the  trypanosomes  to  develop,  the  average 
is  about  4£  days,  yet  tubes  made  from  blood  kept  4  days  developed  in 
4  days.  Furthermore,  whereas  when  the  blood  was  kept  in  the  labo- 
ratory only  2  out  of  8  tubes  developed,  the  trypanosomes  appeared 
on  the  third  and  fourth  days.  The  implication  here  is  that  the 
greater  part  of  the  trypanosomes  in  the  blood  are  either  killed  or  so 
enfeebled  that  they  are  unable  to  develop,  while  the  growth  which 
takes  place  is  due  to  the  hardier  survivors.  This  is  supported  by  the 
character  of  the  growth  which  was  eventually  brought  about  in  the 
tubes.  In  no  case  was  this  luxuriant,  and  it  was  good  hi  only  three 
of  the  tubes,  pointing  to  the  conclusion  that  the  number  of  trypano- 
somes present  in  the  tubes  was  very  much  below  the  average. 

In  ah1  of  the  cases  considered  above,  control  cultures  made  promptly 
after  the  blood  was  drawn  were  positive. 

RESULTS    OBTAINED    WITH    MEASURED    QUANTITIES    OF    BLOOD. 

The  following  table  gives  the  results  obtained  with  measured 
quantities  of  blood,  the  medium  used  being  the  laboratory  make  of 
beef  bouillon,  and  the  cultures  being  made  the  same  day  the  blood 
was  drawn.  Unless  otherwise  stated,  the  culture  tubes  were  kept  in 
the  room.  In  all  cases  given  in  the  table  where  the  quantity  of  blood 
was  small,  and  the  results  negative,  controls  showed  the  blood  used 
to  contain  trypanosomes. 


EXAMINATION    OF    CULTURES. 
Results  obtained  with  measured  quantities  of  blood. 


19 


Culture. 

Date. 

Cow. 

Quantity 
of 
blood. 

Result  (+, 
sositive;—  , 
negative). 

Time. 

4  

1909 
Mar.  29 

688 

c.  c. 
3.0 

+ 

Days. 
»6 

5 

...do  

688 

1.5 

(i) 

31  

Apr.  5 

688 

2.0 

+ 

3 

33            '                  ... 

...do.... 

688 

1.5 

+ 

4 

38 

.  do. 

688 

2.0 

(0 

39  

Apr.  7 

688 

2.5 

+ 

^  3 

41                

...do  

688 

2.0 

+ 

3 

42 

...do.... 

688 

1.5 

(i) 

43..             

...do  

688 

3.0 

+ 

4 

53  

Apr.  12 

688 

5.0 

+ 

3 

64 

.So. 

688 

4.0 

+ 

4 

55 

...do.... 

688 

4.0 

+ 

3 

56 

.do. 

688 

4.0 

+ 

3 

57 

..do.... 

688 

4.0 

+ 

4 

58 

.do. 

688 

3.5 

+ 

3 

59                     

.do  

688 

3.0 

+ 

3 

64  

Apr.  24 

695 

2.0 

+ 

4 

05                      

..do  

695 

1.5 

+ 

66 

.do. 

695 

.4 

+ 

07                         

.do  

695 

1.5 

+ 

08 

.do. 

695 

3.5 

+ 

70.                     

.do.... 

695 

2.5 

+ 

72 

.do.   . 

6% 

3.0 

+ 

73                    

.do.... 

6% 

5.0 

+ 

4 

74 

.do... 

696 

3.5 

75                    

..do  

696 

5.0 

+ 

76 

do.  .. 

6% 

4.0 

77 

do. 

6% 

5.0 

+ 

78 

do. 

696 

3.0 

+ 

79 

do 

696 

4.0 

4- 

81...         

Apr.  20 

697 

2.5 

+ 

4 

82 

do. 

697 

2.0 

+ 

83 

do..  . 

697 

2.0 

+ 

85 

do 

697 

1.0 

4- 

86 

do. 

697 

2.5 

4- 

87 

do. 

697 

2.5 

4- 

88 

do. 

697 

1.0 

+ 

89" 

do 

697 

.5 

4- 

133  

May  13 

688 

2.5 

4- 

3 

154  .                 

.do.  .  .  . 

688 

3.0 

4- 

3 

135  

..do  

688 

2.0 

4- 

3 

136 

do 

688 

">  0 

+ 

137 

do. 

688 

2.5 

4- 

138..                 

.do.  .  . 

lixs 

2.0 

+ 

3 

139  

...do.... 

688 

3.0 

4- 

2 

140..                

.do. 

688 

2.0 

+ 

3 

141 

do. 

688 

1.5 

4- 

142 

do 

688 

1.5 

+ 

144  

...do.... 

688 

2.0 

+ 

3 

145...               

.do.   . 

088 

2.5 

+ 

3 

146  

...do.... 

(>88 

3.0 

+ 

3 

147..          

.do..  .. 

688 

3.0 

+ 

3 

420 

Nov.  17 

218 

1.0 

421 

do 

218 

•>.o 

422 

do. 

218 

3.0 

423 

do. 

218 

4.0 

424 

do. 

218 

5.0 

425  

.do. 

218 

6.0 

+ 

6 

426..               

do. 

218 

7.0 

427  

.do. 

218 

8.0 

+ 

6 

428....               

.do. 

218 

9.0 

+ 

6 

429  

..do. 

218 

10.0 

+ 

6 

436 

do 

218 

1  0 

438 

do. 

218 

3.0 

440 

do 

218 

5  0 

-f- 

441.. 

do. 

218 

0.0 

442 

do 

218 

7  0 

+ 

443.. 

do. 

218 

8.0 

444 

do 

218 

9  0 

445...              

do. 

218 

10.0 

+ 

5 

532  . 

1910 
Apr.  7 

697 

Drops.1 
1 

633 

do 

697 

•> 

550.... 

\pr.  13 

097 

1 

551 

do 

697 

652 

do. 

697 

:i 

868.  ... 

...do... 

097 

I 



i  Culture  in  Incubator. 


1 1  drop=0.0675  c.  c. 


20        TEYPANOSOMA   AMERICANUM,   A   BLOOD  PAKASITE    OF   CATTLE. 
Results  obtained  with  measured  quantities  of  blood — Continued. 


Culture. 

Date. 

Cow. 

Quantity 
of 
blood. 

Result  (+, 
positive;  —  , 
negative). 

Tlme. 

574  

1910 
Apr.  21 

697 

Drops. 
5 

Days. 

575  

...do..  . 

697 

5 

576  . 

.do. 

697 

10 

577  

...do  

697 

10 

618  

July   14 

697 

5 

.f- 

6 

622.... 

do. 

697 

c.  c. 
1  0 

-f 

623.  .. 

...do.  .  . 

697 

1.0 

-(. 

624  

.do. 

697 

1  0 

-1- 

625  

...do... 

697 

1.0 

4- 

Here  it  is  seen  that  the  smallest  quantity  of  blood  to  give  a  positive 
result  was  5  drops,  or  0.3375  c.  c.,  and  that  this  result  was  obtained 
only  once  out  of  three  trials.  Assuming  6,000,000  red  cells  and  10,000 
leucocytes  per  cubic  millimeter,  we  find  as  a  possible  proportion  one 
trypanosome  for  2,022,000,000  red  cells  and  3,370,000  whites.  Hence 
to  find  the  trypanosome  in  the  circulating  blood  would  be  merely  a 
piece  of  good  fortune.  Further,  culture  444,  containing  9  c.  c.  of 
blood,  was  negative,  yet  this  amount  of  blood  contains  90,000,000 
leucocytes. 

For  the  most  part  the  bouillon  used  for  the  cultures  was  that  made 
in  the  laboratory  from  beef,  for  ordinary  bacteriological  work.  Some 
16  tubes  were  used  in  which  the  medium  was  made  from  commercial 
meat  extract.  It  was  not  treated  with  an  alkali,  and  hence  was 
acid  in  reaction.  The  growth  in  the  latter  medium  was  in  general 
not  so  luxuriant  as  that  in  the  laboratory  make  of  bouillon,  and  in 
two  cases  the  trypanosomes  failed  to  appear  at  all,  although  con- 
trols (hi  laboratory  bouillon)  were  positive. 

Fourteen  cultures  were  made  in  physiological  salt  solution,  6  parts 
per  thousand.  In  one  set  of  experiments  with  this  medium  5  c.  c. 
of  salt  solution  was  used  in  each  tube,  to  which  were  added  2,3,4, 
and  5  c.  c.  of  blood,  respectively.  Trypanosomes  never  developed, 
although  hi  all  cases  the  controls  were  positive.  These  results  were 
at  odds  with  those  obtained  by  the  Dudukalovs  (see  page  9). 

In  one  set  of  tubes  cow's  milk  was  used,  the  results  being  negative. 

GENERAL   RESULTS    OF    CULTURAL    WORK. 

The  results  may  be  summarized  as  follows: 

(1)  Trypanosoma  americanum  grows  readily  hi  ordinary  bouillon, 
made  from  either  beef  or  mutton. 

(2)  The  average  time  required  for  the  trypanosomes  to  be  readily 
detected  in  the  tubes  is  3J  days. 


OBSERVATIONS   ON   FRESH   MATERIAL.  21 

(3)  The  smallest  quantity  which  gave  positive  results  was  5  drops, 
or  0.3375  c.   c.     The  largest  measured  quantity  to  give  negative 
results  was  9  c.  c. 

(4)  There  is  a  seasonal  fluctuation,  the  trypanosomes  being  more 
abundant  in  the  spring  and  summer  than  they  are  in  the  autumn. 

OBSERVATIONS  ON  FRESH  MATERIAL. 

The  successful  cultures  were  those  made  by  adding  the  blood  of 
the  cow  to  bouillon  of  several  different  kinds.  The  freshly  made 
culture  necessarily  consisted  of  a  red  column,  composed  of  an  admix- 
ture of  blood  and  the  medium.  Upon  standing  for  about  24  hours 
the  blood  cells  settled  to  the  bottom  of  the  tube,  leaving  above  them 
a  clear  fluid  composed  partly  of  blood  serum  and  partly  of  bouillon 
itself.  At  first  the  surface  of  this  column  was  smooth  and  of  a 
uniform  red,  but  by  the  end  of  the  second  day  it  usually  began  to 
present  appearances  which  were  presumably  caused  by  the  rising  to 
the  surface  of  the  leucocytes.  These  appearances,  however,  were 
very  varied.  In  some  cases  the  surface  remained  perfectly  flat  but 
showed  itself  studded  with  minute  whitish  specks  and  motes  which, 
when  large  enough  for  their  form  to  be  determined,  were  of  irreg- 
ular contours  and  closely  resembled  particles  of  dust.  Frequently, 
however,  a  fuzzy  deposit  appeared  upon  the  surface,  consisting  of  a 
reddish- white  stringy  mass,  which  might  be  elevated  as  much  as 
1  or  2  mm.  These  fuzzy  masses  never  occupied  more  than  a  por- 
tion of  the  surface  of  the  column  of  red  cells.  At  other  times  this 
surface  was  differentiated  into  a  series  of  ripples,  representing  in 
miniature  the  surface  of  wind-blown  sand. 

But  in  spite  of  these  various  aspects,  there  was  never  any  diffi- 
culty in  determining  macroscopically  (or  preferably  with  the  aid  of 
a  hand  lens)  whether  the  tubes  did  or  did  not  contain  trypanosomes, 
at  least  after  they  had  had  time,  if  present,  to  establish  themselves. 
Frequently  by  the  third  day  there  could  be  distinguished  on  the 
surface  of  the  red  cells  minute  dots,  differing  from  the  motes  above 
mentioned  in  that  they  were  white  and  circular.  When  these  had 
reached  a  certain  size  it  could  generally  be  seen  that  their  top  sur- 
faces were  not  flat  but  convex,  and  when  as  sometimes  happened 
they  were  supported  by  the  masses  of  fuzz  and  thus  carried  above 
the  surface  they  showed  as  more  or  less  spherical  bodies.  At  first 
these  trypanosome  colonies  were  very  minute  and  frequently  scat- 
tered over  the  surface  in  a  more  or  less  uniform  manner,  but  as  the 
culture  got  older  they  always  became  larger  and  often  fewer  in 
number.  Increase  in  size  is  of  course  a  matter  of  growth,  and  the 
diminution  in  numbers  was  doubtless  a  case  of  fusion  of  closely 
lying  colonies.  In  mature  cultures  there  were  at  times  but  two  or 
three  colonies — enormous  white  masses  2  or  3  mm.  across. 


22        TBYPANOSOMA   AMERICANUM,   A   BLOOD   PARASITE    OF   CATTLE. 

The  small  colonies  were  always  circular,  the  larger  circular,  oval, 
or  irregular.  A  curved  outline,  however,  was  always  maintained. 
The  form  of  the  colonies,  however,  is  doubtless  merely  a  matter  of 
surface  tension.  Fusion  of  two  very  small  circular  colonies  results 
hi  the  production  of  a  large  circular  one,  but  when  a  certain  size  is 
reached  the  surface  tension  is  able  only  to  impart  to  the  compound 
colony  a  curved  outline.  It  was  at  times  to  be  noted  that  the  sep- 
arate units  composing  the  large  compound  colonies  were  very  incom- 
pletely fused. 

Clearly,  each  separate  colony  which  arose  on  the  surface  of  the 
red  cells  in  a  tube  represented  a  focus  of  infection,  the  later  increase 
in  bulk  being  merely  due  to  the  multiplicative  activity  of  the  tryp- 
anosomes.  No  accurate  counts  were  made,  but  such  foci  probably 
ranged  in  numbers,  in  the  various  tubes,  from  half  a  dozen  or  less  to 
perhaps  100.  These  foci  of  infection  must  in  each  case  be  either  a 
single  trypanosome,  or  some  element  not  a  trypanosome  but  which 
evolves  into  one,  or  two  or  more  trypanosomes  or  such  elements. 

In  the  preliminary  notice  (Bulletin  119)  it  was  stated  that  it  was 
not  possible  to  say  Avhat  this  was,  that  is,  whether  the  element  in 
the  circulating  blood  of  the  cow  was  an  actual  trypanosome  or  some- 
thing of  an  entirely  different  facies,  and  unfortunately  such  observa- 
tions as  have  been  made  since  do  not  settle  this  point  absolutely, 
yet  they  leave  little  doubt  resting  upon  it.  Trypanosomes  were 
never  seen  in  fresh  blood  from  the  cow,  but  they  finally  were  found 
in  stained  smears  of  centrifuged  blood,  the  smears  having  been  made 
3  or  4  hours  after  the  blood  was  drawn.  It  may  be  stated  here  that 
these  organisms  did  not  occur  singly,  but  in  clusters,  and  were 
evidently  in  a  process  of  rapid  multiplication.  The  probability  that 
they  arose  from  the  division  of  a  trypanosome,  and  not  from  some- 
thing else,  is  so  great  as  to  be  a  practical  certainty.  The  work  of 
Wrublewski  and  Behn,  previously  quoted,  supports  this  hypothesis. 
In  that  of  the  former,  the  trypanosomes  described  as  parasites  of 
the  aurochs  are  so  like  T.  americanum  that  the  latter  might  be 
regarded  as  little  more  than  a  variety  of  the  former.  The  failure 
to  find  the  trypanosome  in  the  perfectly  fresh  blood  of  American 
cattle  is  of  little  moment,  since  if  the  organism  is  only  a  variety  of 
T.  wniblewski  it  is  in  a  strange  host,  and  hence  might  be  able  to 
maintain  itself  in  only  very  limited  numbers.  Moreover,  it  is  a 
matter  of  common  knowledge  that  trypanosomes  may  exist  in  the 
blood  of  annuals  in  such  small  numbers  as  to  evade  observation  with 
the  microscope.  A  great  many  such  cases  have  been  brought  out  hi 
the  experimental  work  on  the  pathogenic  trypanosomes  of  mammals, 
and  it  seems  to  be  almost  the  rule  for  those  of  birds.  Evidently, 
when  a  trypanosome  can  not  be  found  in  the  blood  with  the  micro- 
scope, it  is  more  or  less  of  a  venture  to  claim  that  it  exists  there  as 


OBSERVATIONS   ON  FRESH    MATERIAL.  23 

a  trypanosome  and  not  as  something  else.  Yet  in  the  case  of  birds 
it  is  known  that  after  failure  to  find  them  in  the  circulating  blood 
they  have  been  picked  up  in  extremely  small  numbers  in  the  bone 
marrow.  Besides,  in  spite  of  the  enormous  amount  of  work  which 
has  been  done  on  these  flagellates,  nothing  has  ever  been  discovered 
hi  the  blood  but  an  element  instantly  recognizable  as  a  trypanosome. 
Finally,  there  is  Behn's  discovery.  But  this  trypanosome  is  very 
different  from  either  71  americanum  or  T.  wrublewski.  It  is  not  only 
a  very  much  broader  animal,  but  differs  in  that  the  nuclei  are  farther 
back  in  the  body.  It  is  quite  possible,  however,  that  this  trypano- 
some, although  the  forerunner  of  the  organisms  found  in  the  culture 
tubes  by  the  German  investigators,  is  a  wholly  different  species 
from  T.  americanum.  So  far  the  cultural  trypanosome  of  German 
cattle  has  neither  been  described  nor  figured,  and  it  is  hence  impos- 
sible to  compare  it  with  that  from  American  cattle. 

ATTEMPTS     TO     DISCOVER     THE     TRYPANOSOME     IN     FRESHLY     DRAWN 

BLOOD. 

The  experiments  made  at  Washington  to  determine  this  point 
may  now  be  given. 

Freshly  drawn  blood  from  cattle  was  centrifuged,  and  preparations 
were  thereby  obtained  in  which  the  leucocytes  were  as  abundant  as 
the  red  cells.  This  work  was  done  in  June,  July,  and  August,  1910, 
when  the  cultural  method  showed  the  trypanosomes  to  be  abundant. 
Six  different  animals  were  used,  of  which  four  were  healthy,  one  was 
in  an  advanced  stage  of  tuberculosis,  and  one  was  suffering  from  a 
mild  case  of  Texas  fever.1 

A  large  number  of  fresh  preparations  were  examined,  but  nothing 
in  the  way  of  a  trypanosome  was  ever  found.  In  a  search  of  this 
sort  the  examination  of  fresh  material  was  then  and  still  is  believed 
to  be  the  most  efficient  method,  since  the  flagellate  readily  betrays 
its  presence  by  its  motility.  The  work  was  done  with  a  16  mm. 
objective  and  a  No.  12  eyepiece,  and  a  preparation  containing  as 
much  blood  as  a  large  smear  could  be  searched  in  a  few  minutes. 
A  thorough  test  of  this  procedure  led  to  the  view  that  the  trypano- 
somes could  not  be  found  in  recently  drawn  blood,  and  the  examina- 
tion of  stained  smears  was  undertaken  rather  with  the  idea  of  looking 
for  hypothetical  ' ' preflagellate  "  stages  than  for  actual  trypanosomes. 
Yet  it  was  in  these  that  the  discovery  was  made.  The  trypanosomes 
were  found  on  two  slides,  a  small  group  in  each  case,  out  of  some 
25  examined. 

Cultures  were  also  centrifuged.  The  first  experiment,  made  July 
15,  1910,  with  a  culture  23  to  24  hours  old,  revealed  trypanosomes, 

i  This  cow  was  tested  In  October. 


24        TBYPANOSOMA   AMERICANUM,    A    BLOOD   PABASITE    OF    CATTLE. 

singly  or  in  clusters  and  either  associated  or  not  with  white  cells. 
Later  experiments,  made  in  August,  reduced  the  time  to  15  hours. 
Younger  cultures  were  not  tried. 

The  findings,  however,  were  of  considerable  interest.  In  the 
15-hour  culture  only  a  single  trypanosome  was  found,  but  this  was 
an  organism  having  a  body  quite  20  microns  in  length,  and  a  flagel- 
lum  as  long  as  the  body,  making  it  all  of  40  microns  long.  The 
undulating  membrane  was  short,  and  the  appearance  was  typical 
for  Typanosoma  americanum. 

In  a  culture  18^  hours  old  made  from  blood  5^  hours  after  it  was 
drawn  there  were  found  several  clusters  of  trypanosomes,  one  of 
which  must  have  been  composed  of  at  least  75  individuals.  Since  hi 
the  culture  tubes  division  of  the  flagellates  is  by  no  means  a  rapid 
process,  requiring  from  1  to  2  hours,  multiplicative  activity  must 
have  been  going  on  for  some  time.  At  the  slower  rate,  it  would  take 
14  hours  for  one  trypanosome  to  produce  64.  The  organisms  found 
on  the  first  day  in  centrifuged  cultures  differed  in  no  respect,  so  far  as 
could  be  determined,  from  those  found  in  two  and  three  day  cultures, 
studied  without  centrifuging. 

EVOLUTION   OF   THE    TRYPANOSOMES    IN    CULTURE. 

The  history  of  the  evolution  of  the  trypanosomes,  as  it  takes  place 
in  a  successful  culture,  may  now  be  traced. 

As  already  stated,  there  is  a  certain  variability  in  the  rate  of 
evolution,  but  it  may  conveniently  be  considered  under  three  head- 
ings— the  period  of  growth,  of  culmination,  and  of  decline — although 
the  three  processes  to  a  certain  extent  overlap. 

In  cultures  2  to  3  days  old  there  are  present  a  small  number  of 
organisms,  say  from  1  to  6  to  each  preparation,  a  preparation 
probably  containing  1  cubic  millimeter  of  the  culture.  The  animals 
are  ordinarily  short  and  relatively  broad,  with  the  anterior  half 
broader  than  the  posterior.  At  times,  however,  they  are  quite  slender. 
A  flagellum  is  always  present  and  in  most  cases  a  short  undulating 
membrane  can  easily  be  seen.  The  cytoplasm  is  hyaline.  With 
ordinary  light,  granules  can  not  as  a  rule  be  seen,  but  dark-field 
illumination  shows  them  to  be  present.  It  may  also  here  be  noted 
that  this  method  revealed  the  fact  that  in  the  adult  trypanosomes 
the  flagella  often  show  a  double  contour,  even  to  the  extreme  tip. 
The  organisms  are  either  solitaiy,  in  pairs,  or  in  clusters.  The 
clusters  are  nearly  always  of  few  individuals,  but  this  of  course 
depends  upon  the  rate  with  which  multiplicative  activity  has  been 
inaugurated.  The  flagellum  and  undulating  membrane  are  in 
constant  action,  and  progression  in  a  slow,  unsteady  manner,  with 
numerous  changes  of  direction,  is  frequent. 


OBSERVATIONS   ON   FBESH   MATERIAL.  25 

Cultures  3  to  4  days  old  show  all  of  the  phenomena  presented 
by  the  earlier  cultures.  There  are  very  many  more  trypanosomes 
present,  however,  and  the  clusters  average  much  larger.  Solitary 
forms  are  relatively  much  rarer,  almost  all  of  the  annuals  being 
in  the  clusters.  The  trypanosomes  themselves  have  become  longer. 
The  flagella  and  undulating  membranes  are  in  constant  motion, 
and  it  may  here  be  stated  that  motility  does  not  seem  to  fluctuate 
with  age,  senile  and  youthful  forms  showing  it  in  approximately 
the  same  degree. 

Evolution  now  follows  the  lines  already  indicated.  In  the  4 
and  5  day  cultures  solitary  forms  occur,  but  are  scarce.  Nearly 
all  the  trypanosomes  present  are  in  clusters,  which  may  consist  of 
3  to  4  individuals,  or  be  of  enormous  size.  One,  from  a  4-day 
tube,  measured  156  by  190  microns,  and  must  have  been  composed 
of  thousands  of  trypanosomes.  The  individual  animals  are  larger; 
some  long  and  slender,  or  crithidia-like ;  others  show  a  typical 
trypanosome  contour,  while  still  others  have  become  club-shaped. 
Large,  stout  divisional  forms  are  frequent.  In  many  the  cytoplasm 
has  become  very  granular. 

From  the  fifth  and  sixth  days  onward  the  cultures  begin  to  present 
a  far  more  diversified  appearance.  There  are  present  large  and 
small  clusters  in  which  the  trypanosomes  may  be  Jband-shaped  or 
club-shaped.  There  are  many  more  free  individuals,  doubtless 
those  which  have  become  separated  from  the  clusters.  The  free 
forms  are  band-shaped,  club-shaped,  or  short  oval  bodies.  Any  and 
all  of  these  may  be  in  division.  There  also  appear  in  the  older 
cultures  very  large  bodies  with  two  or  more  flagella,  no  doubt  result- 
ing from  a  more  or  less  abnormal  division  process.  The  club-shaped 
forms  and  the  giant  trypanosomes  are  marks  of  degeneracy,  and  as 
the  culture  ages  more  and  more  of  the  trypanosomes  begin  to  undergo 
senile  decay,  and  the  culture  eventually  dies  out. 

No  accurate  data  were  kept  as  to  how  long  the  cultures  generally 
lasted,  but  by  the  end  of  a  month  they  are  very  evidently  degenerate. 
In  one  case  a  tube  charged  63  days  previously  was  observed  to 
show,  microscopically,  a  large  number  of  apparent  colonies.  A  study 
of  these  colonies  under  the  microscope  showed,  however,  that  they 
consisted  merely  of  great  masses  of  granular  balls,  with  here  and 
there  a  feebly  motile,  misshapen,  and  intensely  granular  trypanosome. 

The  process  of  evolution  of  the  individual,  or  of  evolution  com- 
bined with  so-called  involution,  is  not,  except  at  the  outset,  difficult 
to  follow.  As  given  above,  a  solitary  trypanosome  having  a  total 
length  of  40  microns  was  found  in  a  centrifuged  culture  15  hours  old. 
(In  the  2  and  3  day  cultures  the  trypanosomes  are  not  so  large 
as  this.)  It  will  probably  not  be  far  from  the  mark  to  look  upon  a 
trypanosome  of  this  size  as  the  originator  of  the  clusters  which  appear 


26        TRYPANOSOMA  AMERICANUM,   A   BLOOD  PARASITE    OF   CATTLE. 

in  the  tubes.  Division  is  always  by  longitudinal  splitting,  but  may 
be  equal  or  unequal.  The  observations  rather  favor  the  view  that 
it  is  at  first  unequal,  the  original  mother  cell  giving  off  a  short, 
relatively  broad  bud.  Such  unequal  divisions  were  seen  in  the 
ordinary  (not  centrifuged)  cultures,  and  were  not  uncommon  in  one 
of  the  centrifuged  cultures  of  the  first  day.  At  all  events,  beginning 
the  series  with  a  short,  broad,  flagellated  element,  the  history  is  as 
follows:  This  element  divides,  and  the  two  daughter  cells  are  also 
at  first  short,  broad  organisms  provided  with  flagella  and  undulating 
membranes.  The  cytoplasm  is  hyaline,  and  fine  granules  are  prob- 
ably always  present.  An  increase  in  length  produces  a  relatively 
more  slender  form,  which,  in  its  turn,  becomes  a  band-shaped 
typical  trypanosome.  In  tune,  the  anterior  half  becomes  broader 
and  rounded,  like  the  head  of  a  club,  the  posterior  half  remaining 
narrow.  The  flagellum  extends  forward  from  the  anterior  tip  of 
the  broad  anterior  half  and  carries  with  it  a  narrow  band  of 
cytoplasm,  which  is  probably  composed  partly  of  endosarc,  since 
it  is  frequently  granular.  The  narrow  posterior  end  becomes 
narrower;  it  is  reduced  to  a  mere  spike,  and  finally  disappears, 
leaving  the  monadine  forms  which  show  a  flagellum  either  extending 
free  or  accompanied  for  a  part  of  its  length  by  a  band  of  cytoplasm. 
In  certainly  a  great  many  cases,  however,  and  perhaps  all,  before 
the  complete  atrophy  of  the  posterior  end,  the  head  of  the  club  had 
undergone  a  distortion  which  carries  the  point  of  origin  of  the 
flagellum  around  to  one  side  of  the  body,  so  that  forms  are  obtained 
wherein  the  flagellum  extends  out  at  right  angles  to  the  longitudinal 
axis  of  the  body. 

Along  with  the  morphological  changes  there  is  a  conspicuous 
change  in  the  granularity  of  the  cytoplasm.  In  the  young  trypano- 
somes  the  granules  are  small,  but  there  is  a  gradual  increase  hi  size 
and  numbers,  and  the  senile  forms  are  closely  packed  with  coarse 
granules.  The  monadine  form  degenerates  into  a  coarsely  granular 
ball. 

The  genesis  of  the  colonies  takes  place  as  follows:  A  cluster  is 
started,  probably  by  a  single  trypanosome.  This,  by  division, 
produces  a  pair,  joined  by  their  posterior  ends.  A  second  division 
produces  a  group  of  four,  but  the  arrangement  is  not  radial,  but 
linear.  The  four  are  united,  in  common,  by  their  posterior  ends, 
but  one  of  the  pairs  is  slightly  in  advance  of  the  other.  In  further 
divisions  this  linear  arrangement  is  maintained,  so  that  the  clusters 
do  not  consist  of  rosettes,  but  of  strings.  Since,  however,  any  given 
pair  of  trypanosomes  is  very  nearly  at  the  same  level  as  the  pair 
next  to  it,  the  linear  disposition  is  more  or  less  completely  masked, 
and  rounded  clusters  are  produced.  The  actual  arrangement  may, 
however,  be  nicely  demonstrated  by  observing  a  rounded  cluster 


OBSERVATIONS   ON  FRESH   MATERIAL.  27 

by  dark-field  illumination.  As  the  cluster  becomes  heated  by  the 
rays  it  spreads  out,  becoming  larger  and  looser,  and  then  it  can  be 
easily  seen  that  such  a  cluster  consists  of  a  number  of  closely  related 
strings  of  trypanosomes.  It  may  also  be  seen  when  trypanosomes 
have  multiplied  in  a  sealed  preparation  on  a  slide.  Here,  if  the 
blood  film  be  sufficiently  thin,  the  groups  are  constrained  to  develop 
within  one  plane,  and  the  linear  arrangement  is  often  very  plain. 
Accordingly,  the  rounded  cluster  is  a  group  of  the  second  order, 
made  up  by  the  strings,  which  are  in  their  turn  made  up  of  individual 
trypanosomes.  Occasionally  such  strings  (or  perhaps  a  better 
term  is  bouquets)  of  trypanosomes  were  seen  which  were  bipolar; 
that  is,  there  were  two  strings  extending  from  a  central  point  in 
opposite  directions.  The  strings  may  possibly  also  give  off  lateral 
branches. 

In  view  of  the  organization  of  the  clusters,  as  just  indicated, 
there  is  no  objection  to  the  view  that  each  has  arisen  from  a  single 
trypanosome.  If  this  be  so,  we  should  get,  as  a  very  rough  average, 
say  25  to  50  trypanosomes  per  culture  tube,  or  perhaps  10  per  cubic 
centimeter  of  blood.  This  gives  one  trypanosome  to  1,000,000 
white  cells.  The  data  given  on  page  20  show,  however,  that  the 
trypanosomes  are  not  so  abundant  as  this.  Such  figures  are  little 
more  than  guesses,  however,  although  they  show  that  the  trypano- 
somes are  very  rare  in  the  circulating  blood  of  the  cow. 

It  may  be  of  interest  to  give  certain  of  the  observations  in  detail: 
Fresh  preparations  from  culture  tubes  2  to  3  days  old,  examined 
under  the  microscope,  show  in  abundance  clumps  composed  partly 
of  red  cells  and  partly  of  leucocytes.  These  clumps  stand  out  quite 
sharply  from  the  film  of  red  cells  and  evidently  possess  a  considerable 
degree  of  consistency.  Their  production  is  probably  in  part  due  to 
the  fact  that  as  the  leucocytes  rise  to  the  surface  they  tend  to 
entangle  a  certain  number  of  red  cells;  but  there  seems  to  be  some 
other  influence  at  work,  because  at  times  these  clumps  consisted 
entirely  of  red  cells,  and  the  red  cells  were  always  in  the  majority. 
In  all  cases  the  red  cells,  as  a  result  of  mutual  pressure,  were  more 
or  less  misshapen. 

It  was  very  quickly  learned  that  the  place  to  look  for  the  first 
trypanosomes  was  in  these  clumps.  In  the  early  cultures  one  or  two 
trypanosomes  could  usually  be  found  in  association  with  one  of  them, 
either  in  the  midst  of  the  blood  cells  or  lying  at  the  periphery,  in  the 
narrow  clear  space  which  usually  separated  a  clump  from  the  sur- 
rounding film  of  erythrocytes.  A  day  later  there  might  be  a  con- 
siderable number  of  trypanosomes  in  such  a  situation,  mostly  in 
little  clusters  around  the  edges  of  the  clump.  In  a  number  of  cases, 
where  preparations  were  obtained  showing  trypanosomes  in  associa- 
tion with  the  clumps,  the  position  of  these  was  determined  by  means 


28        TRYPANOSOMA  AMERICANUM,   A   BLOOD   PARASITE   OP    CATTLE. 

of  the  mechanical  stage,  and  the  preparation  was  set  aside.  Examina- 
tions on  the  following  day  always  showed  a  conspicuous  increase 
hi  the  number  of  trypanosomes,  and  it  was  hi  such  cases  as  these 
that  the  growth  of  the  trypanosomes  hi  strings  or  bouquets  was  at 
times  demonstrated. 

The  appearance  of  trypanosomes  in  association  with  these  clumps 
of  blood  cells  suggested  at  once  that  it  was  here  that  the  origin  of 
the  foci  of  .infection  was  to  be  found,  and,  since  the  clumps  were 
small,  that  the  problem  of  whether  it  was  an  actual  trypanosome  or 
some  resting  stage  which  gave  birth  to  the  trypanosomes  in  the 
cultures  could  easily  be  solved.  In  the  endeavor  to  elucidate 
this  point  the  dark-field  illumination  was  at  first  used  and  some 
very  deceptive  appearances  were  noted. 

As  stated  above,  these  clumps  are  composed  largely  or  wholly  of 
red  cells,  the  peripheries  of  which,  with  dark-field  illumination,  show 
as  bright  bands  of  a  quite  appreciable  width,  inclosing  a  space  which 
does  not  reflect  the  light  and  is  accordingly  clear.  Since  the  cells 
are  closely  packed  together,  the  appearance  is  that  of  a  region  marked 
out  with  very  irregular  polygons.  When  the  preparations  studied 
were  from  cultures  in  an  appropriate  stage  of  development,  that  is, 
when  the  trypanosomes  were  still  scarce,  observation  would  at  times 
appear  to  show  the  edges  of  one  of  these  red  cells  to  be  in  motion. 
This  motion  at  once  recalled  that  of  the  flagellum  of  a  trypanosome, 
as  it  indeed  proved  to  be,  for  presently  it  could  be  seen  that  it  was 
not  the  edge  of  the  red  cell  that  was  in  motion,  but  a  thread,  a  flagel- 
lum, and  by  careful  observation  the  flagellum  could  be  traced  to  a 
faintly  appearing  trypanosome.  The  curious  point  about  this  phe- 
nomenon was,  however,  that  although  a  point  might  be  selected  where 
the  most  careful  scrutiny  failed  to  reveal  the  presence  of  a  trypano- 
some, the  serpentine  movement  would  presently  manifest  itself,  and 
then  the  trypanosome  itself  could  be  discerned.  It  was  as  if  the 
animals  sprang  into  being  full  fledged. 

The  mystery  was  solved  by  a  study  of  the  clumps  by  transmitted 
light.  It  is  probably  a  matter  of  common  experience  that  fresh 
preparations  are  not  very  satisfactorily  observed  with  an  oil-immer- 
sion lens  with  daylight.  The  usual  method  was  then  somewhat 
modified.  The  small  arc  lamp  belonging  to  the  dark-field  apparatus 
was  used,  its  h'ght  being  first  passed  through  a  piece  of  "euphos" 
glass,  and  then  through  a  vessel  containing  a  solution  of  methylene 
blue.  Euphos  glass  cuts  out  the  destructive  ultra-violet  rays;  the 
methylene-blue  solution  is  for  the  benefit  of  the  observer's  eyes,  and 
can  be  made  of  whatever  density  desired.  Finally — and  this  is  of 
importance — immersion  oil  must  be  placed  between  the  top  of  the 
condenser  and  the  slide.  This  procedure  permits  the  use  of  the 
highest-power  eyepieces  with  a  2  mm.  apochromatic  lens,  and  the 


OBSERVATIONS  ON  FRESH   MATERIAL.  29 

trypanosomes  may  be  studied  under  powers  of  1,500  to  2,250 
diameters. 

It  was  by  this  means  learned  that  the  trypanosomes  which  ap- 
peared so  mysteriously  were  already  present  in  the  mass  of  red-blood 
cells,  and  had  merely  shifted  their  position.  But,  unfortunately,  no 
light  was  shed  on  their  origin.  In  a  great  many  cases  round  cells, 
the  appearance  of  which  was  not  wholly  that  of  normal  leucocytes, 
were  individually  kept  under  observation  for  several  hours,  but 
nothing  was  ever  seen  to  take  place.  The  first  trypanosomes  ever 
seen  in  preparations  from  any  culture  were  elongated,  flagellated 
organisms. 

In  the  early  cultures  the  trypanosomes  occurred  either  singly  or  in 
groups  of  two  or  three.  As  has  already  been  pointed  out,  the  pairs 
arise  by  division,  but  there  is  another  way  whereby  paired  animals 
and  clusters  of  very  few  might  take  origin.  This  would  be  by  ag- 
glutination. Besides  being  associated  with  the  clump  of  red  and 
white  cells,  the  trypanosomes  are  found  wandering  through  the 
preparation.  They  create  little  or  no  disturbance  amongst  the  red 
cells,  since  they  travel  at  a  slightly  higher  level  and  may  indeed  be 
creeping  on  the  under  surface  of  the  cover  glass.  Their  movements 
are  slow  and  unsteady  and  changes  of  direction  are  frequent.  The  fol- 
lowing detailed  observations  are  typical  of  what  may  be  seen: 

MOVEMENTS    OF     TRYPANOSOMES    IN    CULTURES. 

In  a  culture  two  days  old,  studied  March  5,  1910,  two  young  trypan- 
osomes came  together  at  4  p.  m.,  and  for  55  minutes  what  was  seen 
can  best  be  designated  as  a  wrestling  match  between  the  two.  Move- 
ments of  the  flagella  and  undulating  membranes  were  practically 
continuous,  and  the  two  kept  their  bodies  constantly  curved,  each 
looped  around  the  other.  At  4.55  they  both  straightened  out  and  lay 
side  by  side,  bodies  in  contact,  the  flagella  pointing  in  the  same  direc- 
tion. This  position  was,  however,  maintained  but  for  a  minute  or 
two,  after  which  they  again  bent  their  bodies,  looped  themselves 
together,  and  resumed  the  apparent  struggle.  At  5.30  the  observa- 
tion was  interrupted,  to  be  resumed  at  7.15  p.  m.,  when  the  same 
condition  of  affairs  was  found. 

In  another  case,  in  a  3-day  culture,  two  slightly  more  mature 
flagellates  were  seen  to  come  into  contact  and  to  remain  so  for  several 
hours.  Here,  as  in  the  case  described  above,  the  flagella  and  undu- 
lating membranes  were  in  constant  movement,  and  the  animals  also 
spent  a  part  of  the  time  each  looped  around  the  other.  But,  in 
addition,  they  would  frequently  partly  separate  and  then  each,  witli 
straight  longitudinal  axis,  appose  its  body  to  that  of  the  other. 
When  so  disposed,  they  were  oriented  indifferently  in  the  same  or 


30        TRYPANOSOMA  AMERICANUM,   A   BLOOD  PARASITE   OF   CATTLE. 

opposite  directions,  and  sometimes  the  end  of  one  was  opposite  the 
middle  of  the  body  of  the  other.  Under  such  circumstances  it  was 
generally  quite  impossible,  even  with  the  very  favorable  conditions 
under  which  the  observations  were  made,  to  trace  any  line  of  demarca- 
tion between  the  two;  but  neither  here  nor  in  any  other  of  the  cases 
observed  was  there  any  reason  to  suspect  conjugation.  Neither 
is  it  believed  that  unions  of  this  sort  were  permanent,  since  contact 
between  the  two  was  never  by  any  specified  portion  of  the  body,  and 
might  be  instantaneously  broken,  whereas  the  pairs  arising  by  divi- 
sion remained  attached,  and  always  by  their  posterior  ends. 

When  the  trypanosomes  were  not  rare  in  the  mount,  it  was  not  at 
all  unusual  for  a  solitary  individual  to  leave  the  edges  of  the  cell 
clumps  and  wander  alone  in  the  preparation.  There  might  be  quite 
a  few  trypanosomes  wandering  through  the  preparation,  but  there 
was  never  any  evidence  of  any  influence  exerted  at  a  distance.  But 
in  a  number  of  cases  it  was  noted  that  as  soon  as  two  individuals  came 
into  contact  each  at  once  became  more  energetic. 

THE  PROCESS  OF  MULTIPLICATION. 

The  actual  process  of  multiplication  was  not  seen  many  times,  but 
the  following  observation  probably  epitomizes  the  process : 

In  a  4-day  culture,  at  2  p.  m.,  March  7,  1910,  there  were  found  a 
large  and  a  small  trypanosome  in  contact.  The  former  was  a  wholly 
normal  animal  of  adult  aspect,  the  latter  egg-shaped  but  with  a 
pointed  posterior  end  and  a  short  flagellum.  It  was  rather  less  than 
half  as  long  as  the  adult  animal,  and  lay  in  contact  with  the  anterior 
hah*  of  it.  In  a  very  few  minutes  it  was  seen  that  the  flagellum  of  the 
egg-shaped  element  was  much  longer,  and  a  little  later  the  element 
itself  was  seen  to  be  split  halfway  from  the  anterior  to  the  posterior 
end.  Unless  the  appearance  of  singleness  of  this  body  when  first  seen 
was  deceptive,  and  due  to  the  angle  from  which  it  was  observed,  the 
division  into  a  partly  separated  element,  with  the  two  anterior  ends 
each  provided  with  a  flagellum,  did  not  require  more  than  15  or  20 
minutes. 

At  2.35  p.  m.  the  large  trypanosome  broke  away  from  the  paired 
element,  but  some  10  minutes  later  joined  it  again,  wrapping  its 
body  around  the  pair  in  such  a  way  that  there  was  obtained  an  oval 
body  provided  with  three  flagella  at  one  end.  The  trio  remained  in 
intimate  contact  until  4.30  p.  m.,  and  during  all  of  this  time  it  was 
possible  only  momentarily  to  see  any  line  of  demarcation  between  the 
three  elements  composing  it.  At  4.30  the  large  trypanosome  again 
separated  from  the  pair,  and  then  it  was  possible  to  see  that  although 
the  two  small  cells  were  still  in  contact  by  their  posterior  ends,  they 
had  increased  very  considerably  in  length,  and  were  now  much  like 
the  young  forms  seen  in  the  young  cultures. 


OBSERVATIONS   ON   FRESH    MATERIAL.  31 

When  the  original  pair  was  first  seen  it  was  taken  to  be  a  case  of 
unequal  division,  but  the  subsequent  behavior  of  the  large  trypano- 
some  is  rather  against  such  an  interpretation.  The  little  trypano- 
somes, in  which  the  body  is  oval,  and  may  or  may  not  be  pointed  at  the 
ends,  were  frequently  found,  either  singly  or  paired,  in  the  clusters 
occurring  in  the  young  cultures,  and  other  cases  of  division  like  that 
above  described  were  at  times  seen.  In  these,  so  far  as  the  observa- 
tions go,  the  pairs  arising  from  division  remained  in  contact,  but  it  is 
not  impossible  that  separation  may  sometimes  take  place  before 
growth  is  completed,  thus  giving  rise  to  solitary  oval  forms.  It  is 
also  theoretically  possible  that  they  arise  from  unequal  division, 
as  in  Trypanosoma  lewisi,  but  unfortunately  neither  my  preserved 
material  nor  my  observations  on  fresh  material  enable  me  to  settle 
this  point. 

Besides  the  oval  form,  there  was  occasionally  found  in  the  early 
cultures  a  round  element,  about  as  large  as  a  red  blood  cell,  provided 
with  a  long  flagellum.  Such  are  presumably  to  be  regarded  as  modi- 
fications of  the  oval  form. 

MOTZLITY   OF   THE   TRYPANOSOMES. 

Attention  has  been  called  to  the  almost  constant  motility  of  the 
trypanosomes.  This  was,  however,  subject  to  a  certain  variability, 
although  it  was  not  possible  to  determine  upon  what  factors  this 
variability  depended.  The  normal  movement  was  an  undulation  of 
the  flageUum  and  undulating  membrane,  accompanied  by  a  certain 
amount  of  flexion  and  torsion  of  the  anterior  part  of  the  body.  The 
younger  trypanosomes  were  frequently  seen  to  have  their  bodies 
bent  into  circles,  simultaneously  displaying  energetic  movements  of 
the  flagella  and  undulating  membranes.  Such  positions  might  be 
maintained  as  long  as  the  observation  lasted.  But  in  the  fully 
fledged  parasites  the  longitudinal  axis  was  usually  straight  and  the 
postnuclear  part  of  the  body  rigid. 

At  times  trypanosomes,  entangled  amongst  blood  cells  or  granular 
debris,  acted  as  though  seeking  to  get  free.  In  such  cases  periods  of 
violent  activity,  during  which  the  movements  lacked  all  regularity, 
alternated  with  periods  of  complete  rest.  In  the  cases  noted  above, 
where  two  trypanosomes  kept  in  company  for  considerable  periods, 
the  movements  also  lacked  regularity. 

On  one  or  two  occasions,  when  the  temperature  of  the  laboratory 
during  the  night  had  been  unusually  low,  the  trypanosomes  observed 
in  the  morning  seemed  rather  sluggish.  This  observation  suggested 
the  following  experiment:  A  tube  was  placed  hi  a  vessel  containing 
ice  and  salt,  and  kept  at  a  temperature  of  just  above  freezing  for 
nearly  two  hours.  A  preparation  was  then  brought  under  the  mi- 
croscope as  quickly  as  possible,  and  immediately  examined,  with  the 


32        TRYPANOSOMA  AMERICANUM,   A   BLOOD  PARASITE    OF   CATTLE. 


result  that  the  trypanosomes  were  found  to  be  displaying  an  activity 
fully  up  to  normal. 

Nor  is  it  certain  that  heat  increases  motility.  Trypanosomes 
always  become  much  more  active  with  dark-field  illumination,  but 
this  may  be  laid  to  the  ultra-violet  or  Roentgen  rays  rather  than  the 
heat. 

OBSERVATIONS  ON  FIXED  MATERIAL. 

The  observations  made  on  fixed  material  confirm  and  supplement 
those  made  on  fresh  preparations.  In  one  respect,  moreover,  they 

carry  the  study  somewhat  fur- 
ther, since  in  two  cases  smears 
made  from  fresh  blood  show 
trypanosomes  (figs.  1  and  2). 

The  blood  from  which  these 
smears  were  made  was  drawn 
at  the  Bureau  Experiment  Sta- 
tion, sent  into  the  laboratory, 
and  centrifuged  before  the 
preparations  were  made  from 
it.  The  interval  was  probably 
about  four  hours.  Hence  the 
blood  was  not  in  a  strict  sense 
freshly  drawn,  yet  the  observa- 
tion compares  with  many  others 
made  upon  trypanosomes.  In 
many  cases  in  the  literature 
where  trypanosomes  are  de- 
scribed as  present  in  the  circu- 
lating blood,  the  blood  in  which 
they  were  found  had  either  been 
removed  from  the  living  animal 
for  some  time  or  had  even  been 
taken  from  a  dead  animal. 
There  had  therefore  been  time 
for  it  to  cool  to  the  tempera- 
ture of  the  ah-.  A  case  in  point 
is  that  of  Trypanosoma  wrublewski,  found  only  in  blood  from  dead 
bison.  But  in  the  case  in  hand  it  is  believed  that  the  removal  of 
the  trypanosomes  from  the  circulation  had  an  immediate  effect,  for 
the  parasites,  as  seen  in  figures  1  and  2,  are  evidently. in  rapid 
division.  Their  extreme  scarcity  in  the  cow  is  practical  proof  that 
in  such  a  situation  the  multiplicative  energy  is  in  abeyance. 

The  only  stimuli  to  which  the  parasites  in  the  drawn  blood  were 
subjected  were  the  lowering  of  the  temperature,  the  defibrinating  of 


FIG.  1.— Forms  of  Trypanosoma  americanum  from 
centrifuged  blood. 


OBSERVATIONS   ON   FIXED   MATERIAL. 


33 


the  blood,  and  the  mechanical  disturbance  consequent  upon  cen- 
trifuging.  It  is  hardly  likely  that  defibrinating  could  have  any  effect, 
and  the  centrifuging  was  carried  on  for  only  a  few  minutes,  the  prepa- 
rations being  then  made  at  once.  Hence  no  time  was  given  for  the 
number  of  divisions  which  the  figures  show  to  have  taken  place. 
Probably,  then,  it  is  the  lowering  of  the  temperature  which  induces 
multiplication,  although  it  does  not  necessarily  follow  that  this  is  in 
any  way  correlated  with  the  removal  of  the  trypanosomes  by  a  biting 
fly.  The  mere  cooling  itself  might  readily  furnish  all  the  stimulus 
needed. 

MORPHOLOGY   OF  THE   FORMS    IN   THE   BLOOD. 

Taking  up  now  the  trypanosomes  as  they  appeared  in  the  centri- 
fuged  blood,  it  will  be  seen  that  they  are  normal  in  shape,  flagellated, 
and  possess  a  large  tropho- 
nucleus  and  a  conspicuous 
vacuole.  Unfortunately,  the 
stain  used  on  the  two  slides 
on  which  they  occurred  was 
not  in  proper  condition,  and 
stained  the  cytoplasm  so  in- 
tensely that  the  situation  of 
the  kinetonuclei  was  ob- 
scured.1 

For  the  group  shown  in 
figure  1  the  average  size  of  the 
body  was  14.7  by  4  microns. 
In  the  case  of  figure  2  the 
dimensions  were:  Forms  in 
division,  13.9  by  4  microns; 
forms  not  in  division,  16.8  by 
3.6  microns. 

A  selection  of  14  of  the  fig- 
ured flagellates,  confined  to 
those  which  did  not  appear  to 

i  i  ,i          i«    ,  i          FiQ.  2. — Forms  of  Trypanusoma  Americanum  from 

have    been    greatly   distorted  centn/uged  blood. 

in  fixation,  gave   an   average 

measurement  of  16.8  by  3.8  microns.  Newly  born  daughter  cells 
can  not  be  larger  and  are  probably  somewhat  smaller  than  the 
mother  cells  from  which  they  were  derived.  Hence  the  trypano- 
some,  as  it  would  appear  in  the  circulating  blood,  probably  has  a 
length  of  at  least  20  microns  excluding  the  flagellum,  and,  as  noted 
on  page  24,  a  trypanosome  of  this  size  was  found  in  a  culture  of  the 
first  dav. 


1  Efforts  to  improve  the  staining  of  these  two  slides  were  futile.    The  stain  resisted  a  treatment  with  acid 
alcohol  of  sufficient  duration  to  injure  the  trypanosomes  themselves. 


34       TBYPANOSOMA  AMEBICANUM,  A   BLOOD  PABASITE   OF  CATTLE. 
MORPHOLOGY  OF  THE   FORMS   IN   CULTURE. 

Figures  3,  4,  5,  and  6  show  the  appearance  in  smears  from  the 
early  cultures.  The  shape  is  to  a  large  extent  artificial;  for  one 
thing,  the  early  stages  appear  to  contract  on  drying,  becoming 
shorter  and  broader.  Primarily,  however,  the  distortion  is  due  to 
the  fact  that  as  the  spherical  cluster  dries  it  is  fixed  flat,  with  results 
such  as  are  shown  very  plainly  in  figure  5.  The  two  cells  shown  in 
figure  4  are  probably  somewhat  contracted.  In  the  film  they  meas- 
ured 10  microns  long.  In  figure  6  the  largest  trypanosome  has  the 
body  24.7  microns  long.  The  group  of  six,  shown  in  this  figure, 
give  clear  indications  of  the  method  of  their  origin,  and  are  proba- 
bly all  derived  from  a  single  trypanosome.  All  but  one  are  oriented 
in  the  same  direction,  and  it  is  evident  that  this  odd  member  of  the 

group  has  just  been  forced  out  of  the 
**        >5  place  which  it  had  been  occupying. 

Evolution,  as  already  set  forth  on  pages 
24  et  seq.,  results  in  the  production  of 
longer  and  relatively  and  often  actually 
more  slender  flagellates.  Figure  7  from  a 
culture  on  the  fifth  day  shows  two  try- 
panosomes.  They  are  longer  and  some- 
what more  slender  than  those  shown  in 
figures  1  and  2.  Figures  8  and  9  are  from 
cultures  of  the  same  age.  Here  the  ani- 
mals are  long  and  slender,  that  of  figure  8 
measuring  27.8  microns  for  the  body. 

FIG.  3. — Forms  of  Trypanosoma  ameri-       „,,  11,.  t  i  i 

camwn  from  bouillon  culture  96  hours     The   undulating   membrane  also  shows 
old.  The  individual  members  of     mOre  distinctly  in  this  figure.     It  was 

the  cluster  are  distinguished  by  the  ,     ,  .",  i  •    i  i  e 

letters  a  to  i.  such  f  orms  as  these  which  were  spoken  of 

as  crithidial  in  the  preliminary  notice, 

but  so  much  confusion  exists  in  the  literature  as  to  what  precisely 
are  the  characters  of  the  genus  Criflddia  that  the  derived  adjective 
is  perhaps  best  let  alone. 

Figures  11  to  15  show  forms  from  older  cultures.  Evidently  after 
a  certain  maximum  length  is  reached  the  animals  again  become 
broader.  Finally,  by  a  considerable  thickening  of  the  anterior  end, 
the  club-shaped  form  is  produced.  Very  marked,  also,  is  the 
increased  conspicuousness  of  the  undulating  membrane. 

In  figures  13  and  14  are  shown,  among  others,  forms  having  a 
broad,  rounded  anterior  end.  Posteriorly  the  body  is  rigid  and 
tongue-shaped,  and  this  portion  finally  degenerates  and  disappears. 
Anteriorly  the  cytoplasm  extends  out  in  a 'long  tapering  portion, 
very  flexible  and  mobile  in  the  living  animal  and  bearing  the  undu- 
lating membrane.  This  portion  also  becomes  absorbed  in  the  gen- 


OBSERVATIONS   ON   FIXED   MATERIAL. 


35 


esis  of  the  monadine  forms,  and  trypanosomes  having  this  outline 
are  doubtless  more  or  less  senile. 

Figure  12  shows  the  largest  individual  found  in  the  preparations, 
the  body  alone  measuring  38  microns. 

CYTOLOGY   OF   THE   FORMS    IN    CUI/TUHE. 

Along  with  the  changes  in  size  and  shape  there  are  conspicuous 
cytological  changes.  With  reference  to  these,  however,  it  is  desir- 
able to  say  that  the  material  used  was  all  "dry,"  and  all  stained 


FIGS.  4  to  9.— Forms  of  Trypanosoma  americanum  from  bouillon  cultures  112  hours  old. 

with  Wright's  stain.  Of  late  a  good  deal  of  the  work  on  trypano- 
somes has  been  done  with  material  fixed  by  the  "wet"  method,  and 
the  dry  method  has  been  extensively  criticized.  But  the  use  of 
Wright's  stain  on  dried  blood  is  a  standard  procedure,  and  the  results 
are  so  evidently  accurate  for  such  delicate  structures  as  leucocytes 
that  they  can  scarcely  be  very  far  wrong  for  trypanosomes. 

In  the  trypanosomes  from  the  centrifuged  blood  of  the  cow  the 
flagella  stained,  but  poorly.  In  the  early  cultures,  2,  3,  and  4  days, 
they  stain,  but  often  very  faintly,  this  peculiarity  being  the  cause  of 
the  error  made  in  the  preliminary  notice.  Later,  however,  their 


36        TRYPANOSOMA   AMERICANUM,   A   BLOOD   PABASITE    OF   CATTLE. 

affinity  for  the  stain  becomes  much  greater,  and  in  the  fully  fledged 
animals  the  flagella  are  conspicuous.  The  color  is  always  red.  In 
nearly  all  cases  the  tip  is  thickened  to  form  a  minute  knob,  as  in 
Trypanosoma  wrublewski.  In  the  newly  born  forms  the  flagellum  is 
short;  in  the  adults  it  is  long;  probably  on  the  average  it  is  as  long 
as  the  body.  Frequently,  however,  in  the  older  cultures,  the  flagel- 
lum is  two  or  even  three  times  longer  than  the  body. 

In  fresh  preparations  an  undulating  membrane  is  always  in  evi- 
dence, but  in  stained  material  made  from  young  cultures  it  can  not 
as  a  rule  be  made  out.  Later  it  broadens  and  becomes  conspicuous, 
but  is  always  short. 

The  kinetonucleus  from  the  first  to  the  last  stains  intensely, 
usually  a  deep  garnet  color.  It  is  sometimes  round,  more  usually 
oval,  and  appears  merely  to  constrict  into  two  at  the  time  of  division. 
It  seems  always  to  be  associated  with  a  vacuole,  which  is  at  times 
as  large  as  the  trophonucleus.  In  cultures  2,  3,  and  4  days  old, 
when  the  trypanosomes  are  in  rapid  division,  this  vacuole  is  obvi- 
ously merely  a  cavity  within  the  cytoplasm. 
It  is  of  irregular  outlines,  sometimes  lobu- 
lated,  sometimes  almost  tubular.  It  presents 
the  peculiarity  that  in  these  early  stages  of 
evolution  it  appears  to  be  open  to  the  exterior. 
The  appearance  presented  recalls  what  is  found 
in  Euglena.  Some  euglenoid  type  might  easily 
have  been  the  ancestor  of  the  Trypanosomidse, 
in  which  case  this  vacuole  might  be  regarded 
as  the  rudiment  of  a  cytopharynx.  The  data 
presented,  however,  are  obviously  insufficient 
to  warrant  doing  more  than  putting  forward  the  above  as  a  possible 
suggestion. 

Later,  the  vacuole  becomes  round  or  oval,  ceases  to  give  the 
appearance  of  being  connected  with  the  exterior,  and  furthermore 
absorbs  the  stain,  becoming  pink.  Although  in  adult  trypanosomes 
it  frequently  can  not  be  demonstrated,  it  is  perhaps  a  constant  fea- 
ture and  has  been  found  in  the  monadine  forms,  the  end  terms  of 
the  series. 

As  well  as  could  be  made  out  in  the  trypanosomes  from  the  freshly 
drawn  blood,  the  trophonucleus  consisted  of  a  large,  tense  sac,  and 
such  is  clearly  its  condition  in  animals  from  the  young  cultures.  It 
stains  homogeneously  and  so  faintly  that  the  actual  color  is  often 
difficult  to  determine;  generally  is  of  a  reddish  cast.  In  the  very 
early  cultures — 2  and  3  days — the  trophonucleus  is  very  conspicu- 
ous, contrasting  sharply  with  the  blue  cytoplasm.  Later  it  becomes 
less  homogeneous,  presenting  a  ground  substance  in  which  is  a 
quantity  of  amorphous  matter  having  a  different  staining  reaction. 


OBSERVATIONS   ON   FIXED   MATERIAL. 


37 


In  the  late  stages  the  appearance  is  that  of  the  conventional  tryp- 
anosome  nucleus,  an  oval  body  provided  with  a  number  of  large, 
rounded  granules.  The  appearances,  however,  are  manifold,  and 
have  not  been  worked  out  in  detail.  Presumably,  two  factors  are 
at  work — the  age  of  the  trypanosome  and  its  state  with  reference 
to  division — and  each  of  these  is  superposed  upon  the  other. 


11 


12 


FIGS.  11  and  12.— Forms  of  Trypanosoma  americanum  from  bouil- 
lon cultures  6  days  old. 

Flos.  13, 14,  and  15. — Forms  of  Trypanosoma  americanum  from 
bouillon  10  days  old. 

The  cytoplasm  hi  the  early  stages  stains  solidly,  but  is  denser 
in  the  middle  of  the  body  than  at  the  ends.  It  is  always  a  clear  blue. 
For  the  first  several  days  it  appears  to  be  provided  with  a  considerable 
number  of  small  vacuoles  of  uniform  size.  Since,  however,  observa- 
tions on  fresh  material  show  these  young  trypanosomes  to  contain 
granules,  and  since  there  is  no  differentiation  of  the  cytoplasm  in  the 
fixed  preparations  other  than  these  apparent  vacuoles,  the  probability 
is  that  they  are  really  granules  which  refuse  the  stain.  This  sup- 
position receives  support  from  the  fact  that  the  granules  of  leuco- 


38       TEYPANOSOMA  AMERICANUM,  A  BIXX)D  PARASITE   OF  CATTLE. 

cytes  at  times  appear  as  holes  in  the  stained  material.  Later,  the 
cytoplasm  loses  its  solidity  and  homogeneity  and  conspicuous  blue 
and  violet  granules  become  abundant,  and  in  old  cultures  the 
trypanosomes  frequently  consist  of  little  else  than  sacs  more  or  less 
completely  filled  with  coarse  granules. 

PRINCIPAL  CHARACTERISTICS  OF  TRYPANOSOMA  AMERICANUM. 

Trypanosoma  americanum  is  a  large  try panosome .  Figure  1 2  shows 
the  largest  specimen  found,  the  body  measuring  some  38  microns  in 
length.  In  this  particular  case  the  flagellum  was  either  short  or  else 
failed  to  stain  throughout.  But  since  the  flagellum  is  normally  as 
long  as  the  body,  a  total  length  of  75  microns  is  by  no  means  unlikely. 

The  most  marked  peculiarity,  however,  is  the  very  short  undulating 
membrane.  The  kinetonucleus  may  be  in  front  of,  alongside,  or 
behind  the  trophonucleus,  and  the  two  are  always  close  together. 
But  the  reason  for  the  shortness  of  the  undulating  membrane  is  that 
the  nuclear  system  is  pushed  forward.  The  usual  situation  for  the 
trophonucleus  of  a  trypanosome  is  near  the  middle  of  the  body, 
but  in  T.  americanum  it  is  at  the  junction  of  the  anterior  and  middle 
thirds.  That  is,  the  distance  from  the  middle  part  of  the  tropho- 
nucleus to  the  anterior  end  of  the  trypanosome  averages  33  per  cent 
of  the  body  length,  and  in  the  specimens  measured  ranged  from 
25  to  40  per  cent.  In  this  respect  T.  americanum  differs  from  the 
T.  transvaliense  phase  of  T.  iheileri,  in  which,  according  to  the  data 
given  by  Luhs,  this  distance  is  about  50  per  cent  of  the  whole.  Here 
again  it  agrees  with  T.  wrublewski,  although  even  in  the  latter  the 
nucleus,  according  to  the  published  figures,  is  hardly  so  far  forward. 

CONCLUSIONS. 

1.  Trypanosoma  americanum,  lives,  ha  all  probability  as  a  typical 
trypanosome,  in  the  blood  of  perhaps  75  per  cent  of  yearling  and 
adult  American  cattle,  but  is  not  present  in  young  calves. 

2.  It  comes  very  close  to  T.  wrublewski  of  the  European  bison, 
and  may  be  only  a  variety  of  that  species. 

3.  Removal  from  the  circulating  blood  stimulates  multiplicative 
energy,  apparently  merely  as  the  result  of  a  cooler  environment. 

4.  Removed  from  the  cow  and  placed  under  appropriate  conditions, 
multiplicative  energy  runs  far  in  advance  of  growth  energy;  hence — 

5.  The  trypanosomes  divide  and  redivide  very  rapidly,  and  hi 
consequence  become  smaller  than  the  blood  forms. 

6.  At  the  end  of  a  few  days,  multiplicative  energy  weakening, 
the  organisms  have  an  opportunity  to  grow  and  to  reach  their 
normal  size. 

7.  The  adults  are  at  first  very  slender,  but  in  time  increase  in 
breadth  and  may  become  very  large. 


CONCLUSIONS — BIBLIOGRAPHY.  39 

8.  As  the  cultures  reach  and  pass  their  maxima  the  individuals 
become   club-shaped   and   eventually   transform   into   rounded   or 
oval  elements,  provided  each  with  a  long  fiagellum. 

9.  Changes  in  the  morphology  of  the  nuclear  system,  and  in  the 
texture   and   chemical   nature   or   composition   of   the   cytoplasm, 
accompany  changes  in  the  facies  of  the  entire  organism. 

10.  A  distinguishing  character  is  the  situation  of  the  trophonucleus, 
which  is  normally  at  the  union  of  the  anterior  and  middle  thirds. 

BIBLIOGRAPHY. 

BEHN,  PAUL. 

19100-. — Uber  Entwicklungsformen  des    Trypanosoma  franki  <Berl.  tierarztl. 

Wchnschr.,  v.  26  (42),  20.  Oct.,  pp.  809-810,  1  fig. 
1910/J. — Praflagellate  Entwicklungsstadien  der  in  deutschen  Rindern  kulturell 

nachweisbaren  Trypanosomen  <Berl.   tierarztl.  Wchnschr.,   v.  26  (46),  17. 

Nov.,  p.  899. 
19107-. —  Infektion  eines  Kalbes  mit  Trypanosomen  vom  Typus  des  Trypanosoma 

theileri  mittelst  Blut  von  Kiihen,  in  denen  nur  kulturell  Flagellaten  nachweis- 

bar  waren  <Berl.  tierarztl.  Wchnschr.,  v.  26  (50),  15.  Dec.,  pp.  998-1000,  figs. 

1-4. 

DELANOE,  P. 

1911. — Presence  de  trypanosomes  chez  les  bovid^s  en  France  <Bull.  Soc.  path, 
exot.,  Par.,  v.  4  (2),  8  fev.,  pp.  112-116. 

DUDUKALOV,  A.;  &  DUDUKALOVA,  N. 

1910. — Ob  iskusstvennom  kultivirovanii  tripanozom  naidennykh  u  rogatago 
skota.  Sooshchenie  vtoroe  <Ark.  vet.  nauk,  S.-Peterb.,  v.  40  (1),  pp.  1-4, 
1  pi.,  figs.  1-16. 

KNUTH,  PAUL. 

1910. — Uber  die  in  deutschen  Rindern  gefundenen  Trypanosomen  <Berl.  tier- 
arztl. Wchnschr.,  v.  26  (42),  20.  Oct.,  pp.  810-811. 

KNUTH,  PAUL;  &  RAUCHBAAR,  GUSTAV. 

1910. — Zum  Vorkommen  von  Trypanosomen  bei  Rindern  in  Deutschland.  Zweite 
vorlaufige  Mitteilung  <Berl.  tierarztl.  Wchnschr.,  v.  26  (31),  4.  Aug.,  pp. 
609-610. 

KNUTH,  PAUL;  RAUCHBAAR,  GUSTAV;  &  MORGENSTERN,  PAUL. 

1910. — Nachweia  von  Trypanosomen  beim  Rinde  im  Kreise  Oberwesterwald 
mittelst  Ziichtung  in  Blut-Bouillon.  Vorlaufige  Mitteilung  <Berl.  tierarztl. 
Wchnschr.,  v.  26  (27),  7.  Juli,  pp.  539-540. 

MARTINI,  ERICH. 

1909. — The  development  of  a  piroplasma  and  trypanosoma  of  cattle  in  artificial 
culture  media  <Philippine  J.  Sc.,  Manila,  v.  4  (3),  June,  pp.  147-169,  pis.  1-6, 
fig.  1-34. 

MlYAJIMA,  M. 

1907. — On  the  cultivation  of  a  bovine  piroplasma:  A  preliminary  communication 
<Philippine  J.  Sc.,  Manila,  B.  Med  Sc.,  v.  2  (2),  May,  pp.  83-92,  pis.  1-3, 
figs.  1-12. 

SCHMITT,  F.  M. 

1910. — Zum  Vorkommen  von  Trypanosomen  vom  Typus  der  Trypanosoma  theileri 
in  deutschen  Rindern  <Berl.  tierarztl.  Wchnschr.,  v.  26  (44),  3.  Nov.,  pp. 
841-842. 

SERGENT,  EDMOND;  &  ETIENNK. 

1911. — Presence  de  trypanosomes  chez  les  bovid6s  en  Algeiie  <Bull.  Soc.  path, 
exot.,  Par.,  v.  4  (1),  11  Jan.,  pp.  40-42. 

STOCKMAN,  S. 

1910. — Preliminary  note  on  a  trypanosome  of  British  cattle  <J.  Comp.  Path.  & 
Therap.,  Edinb.  &  Lond.,  v.  23  (2),  June  30,  pp.  189-192,  1  fig. 

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